Molecules that selectively home to vasculature of  premalignant or malignant lesions of the pancreas and other organs

ABSTRACT

The invention provides a conjugate that includes a therapeutic moiety linked to a peptide or peptidomimetic that selectively homes to vasculature of premalignant pancreas. The peptide or peptidomimetic contains at least 5 contiguous amino acids of an amino acid sequence selected from CRSRKG (SEQ ID NO:9) and CEYQLDVE (SEQ ID NO:34), or a conservative variant or peptidomimetic thereof. The invention additionally provides a conjugate containing a therapeutic moiety linked to a peptide or peptidomimetic that selectively homes to pancreatic tumor cells and pancreatic tumor vasculature, the peptide or peptidomimetic comprising at least 5 contiguous amino acids of an amino acid sequence selected from CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ ID NO:19), and VGVGEWSV (SEQ ID NO:35), or a conservative variant or peptidomimetic thereof.

This application is a continuation of U.S. application Ser. No.10/977,367, filed Oct. 29, 2004, which claims the benefit of U.S.provisional Ser. No. 60/516,118, filed Oct. 31, 2003, each of which theentire contents are incorporated herein by reference.

This invention was made with government support under CA82713, awardedby the National Cancer Institute, and DAMD 17-02-1-0315, awarded by theDepartment of Defense. The government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

A hurdle to advances in preventing and treating cancer is the lack ofagents that effectively target a cancer or pre-cancerous tissue whilesparing normal tissues. Radiation therapy and surgery, which aretypically localized treatments, can cause substantial damage to normaltissue in the treatment field, resulting in scarring and loss of normaltissue. Furthermore, chemotherapy, which is typically a systemictreatment, can cause substantial damage to normal organs such asnon-cancerous skin, bone marrow, mucosa, and small intestine, inparticular because these tissues undergo rapid cell turnover andcontinuous cell division. As a result, undesirable side effects such asnausea, loss of hair and drop in blood cell count can result fromsystemic treatment with a chemotherapeutic agent. Such undesirable sideeffects often limit the amount of drug that can be safely administered,thereby reducing patient survival rates and quality of life.

Selective delivery of therapeutics such as anti-angiogenic agents tovasculature that supports tumors would result in less toxic therapysince rapidly proliferating normal cells would be spared. Similarly,selective delivery of anti-angiogenic agents to vasculature ofpremalignant tissues would provide a prophylactic strategy for reducingthe risk of cancer. However, to date, it has been difficult to producedrugs that are delivered specifically to tumor vasculature or tovasculature of premalignant tissues. Thus, there is a need for moleculesthat selectively target tumor tissues and vasculature, such aspancreatic tumors and vasculature, as well as for molecules thatselectively target premalignant tissues and vasculature, such aspremalignant pancreas and vasculature. The present invention satisfiesthese needs and provides related advantages as well.

2. Background Information

A hurdle to advances in preventing and treating cancer is the lack ofagents that effectively target a cancer or pre-cancerous tissue whilesparing normal tissues. Radiation therapy and surgery, which aretypically localized treatments, can cause substantial damage to normaltissue in the treatment field, resulting in scarring and loss of normaltissue. Furthermore, chemotherapy, which is typically a systemictreatment, can cause substantial damage to normal organs such asnon-cancerous skin, bone marrow, mucosa, and small intestine, inparticular because these tissues undergo rapid cell turnover andcontinuous cell division. As a result, undesirable side effects such asnausea, loss of hair and drop in blood cell count can result fromsystemic treatment with a chemotherapeutic agent. Such undesirable sideeffects often limit the amount of drug that can be safely administered,thereby reducing patient survival rates and quality of life.

Selective delivery of therapeutics such as anti-angiogenic agents tovasculature that supports tumors would result in less toxic therapysince rapidly proliferating normal cells would be spared. Similarly,selective delivery of anti-angiogenic agents to vasculature ofpremalignant tissues would provide a prophylactic strategy for reducingthe risk of cancer. However, to date, it has been difficult to producedrugs that are delivered specifically to tumor vasculature or tovasculature of premalignant tissues. Thus, there is a need for moleculesthat selectively target tumor tissues and vasculature, such aspancreatic tumors and vasculature, as well as for molecules thatselectively target premalignant tissues and vasculature, such aspremalignant pancreas and vasculature. The present invention satisfiesthese needs and provides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides a peptide or peptidomimetic having a length ofless than 60 amino acid residues and containing at least 5 contiguousamino acids of CRGRRST (SEQ ID NO:5), CRSRKG (SEQ ID NO:9), CKAAKNK (SEQID NO:15), CKGAKAR (SEQ ID NO:19), FRVGVADV (SEQ ID NO:27), CEYQLDVE(SEQ ID NO:34) and VGVGEWSV (SEQ ID NO:35), or a conservative variant orpeptidomimetic thereof. In embodiments of the invention, the peptide orpeptidomimetic contains 40 amino acids, 20 amino acids or 10 aminoacids.

The invention provides a conjugate that includes a therapeutic moietylinked to a peptide or peptidomimetic that selectively homes tovasculature of premalignant pancreas. The peptide or peptidomimeticcontains at least 5 contiguous amino acids of an amino acid sequenceselected from CRSRKG (SEQ ID NO:9) and CEYQLDVE (SEQ ID NO:34), or aconservative variant or peptidomimetic thereof. In embodiments of theinvention, the peptide or peptidomimetic has a length of less than 100residues, less than 50 residues and less than 25 residues. Also providedby the invention is a conjugate containing a therapeutic moiety linkedto a peptide or peptidomimetic that selectively homes to vasculature ofpremalignant pancreas, in which the peptide or peptidomimetic bindsspecifically to a cognate receptor for SEQ ID NO:9 or SEQ ID NO:34.

The invention additionally provides a conjugate containing a therapeuticmoiety linked to a peptide or peptidomimetic that selectively homes topancreatic tumor cells and pancreatic tumor vasculature, the peptide orpeptidomimetic comprising at least 5 contiguous amino acids of an aminoacid sequence selected from CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ IDNO:19), and VGVGEWSV (SEQ ID NO:35), or a conservative variant orpeptidomimetic thereof. In embodiments of the invention, the peptide orpeptidomimetic has a length of less than 100 residues, less than 50residues and less than 25 residues. Also provided by the invention is aconjugate that contains therapeutic moiety linked to a peptide orpeptidomimetic that selectively homes to pancreatic tumor cells andpancreatic tumor vasculature, wherein the peptide or peptidomimeticbinds specifically to a cognate receptor for SEQ ID NO:15, SEQ ID NO:19or SEQ ID NO:35.

The invention provides a conjugate that contains a therapeutic moietylinked to a peptide or peptidomimetic that selectively homes topremalignant and malignant pancreatic vasculature, the peptide orpeptidomimetic containing at least 5 contiguous amino acids of an aminoacid sequence selected from CRGRRST (SEQ ID NO:5) and FRVGVADV (SEQ IDNO:27), or a conservative variant or peptidomimetic thereof. Inembodiments of the invention, the peptide or peptidomimetic has a lengthof less than 100 residues, 50 residues and less than 25 residues. Alsoprovided by the invention is a conjugate that contains a therapeuticmoiety linked to a peptide or peptidomimetic that selectively homes topancreatic tumor cells and pancreatic tumor vasculature, wherein thepeptide or peptidomimetic binds specifically to a cognate receptor forSEQ ID NO:5 or SEQ ID NO:27

The conjugates of the invention can be linked to a variety of moieties.In one embodiment, the moiety is a therapeutic moiety. In anotherembodiment, the moiety is a detectable moiety.

The invention provides a multivalent conjugate, containing a therapeuticmoiety linked to at least two peptides or peptidomimetics thatselectively home to vasculature of premalignant pancreas, each of thepeptides or peptidomimetics containing at least 5 contiguous amino acidsof an amino acid sequence selected from CRSRKG (SEQ ID NO:9) andCEYQLDVE (SEQ ID NO:34).

The invention also provides a multivalent conjugate that contains atherapeutic moiety linked to at least two peptides or peptidomimeticsthat selectively home to vasculature of premalignant pancreas, whereineach of the peptides or peptidomimetics binds specifically to a cognatereceptor for SEQ ID NO:9 or SEQ ID NO:34.

The invention further provides a multivalent conjugate that contains atherapeutic moiety linked to at least two peptides or peptidomimeticsthat selectively home to pancreatic tumor cells and pancreatic tumorvasculature, each of the peptides or peptidomimetics containing at least5 contiguous amino acids of an amino acid sequence selected from CKAAKNK(SEQ ID NO:15), CKGAKAR (SEQ ID NO:19), and VGVGEWSV (SEQ ID NO:35), ora conservative variant or peptidomimetic thereof.

Also provided by the invention is a multivalent conjugate that containsa therapeutic moiety linked to at least two peptides or peptidomimeticsthat selectively home to premalignant and malignant pancreaticvasculature, wherein each of the peptides or peptidomimetics bindsspecifically to a cognate receptor for an amino acid sequence selectedfrom CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ ID NO:19), and VGVGEWSV (SEQID NO:35).

The invention provides a multivalent conjugate that contains atherapeutic moiety linked to at least two peptides or peptidomimeticsthat selectively home to premalignant and malignant vasculature, each ofthe peptides or peptidomimetics containing at least 5 contiguous aminoacids of an amino acid sequence selected from CRGRRST (SEQ ID NO:5) andFRVGVADV (SEQ ID NO:27), or a conservative variant or peptidomimeticthereof.

Also provided is a multivalent conjugate that contains a therapeuticmoiety linked to at least two peptides or peptidomimetics thatselectively home to pancreatic tumor cells and pancreatic tumorvasculature, wherein each of the peptides or peptidomimetics bindsspecifically to a cognate receptor for an amino acid sequence selectedfrom CRGRRST (SEQ ID NO:5) and FRVGVADV (SEQ ID NO:27).

The invention provides a method of directing a moiety to a pancreaticpremalignant lesion in an individual. The method involves administeringto the individual a conjugate containing a moiety linked to (a) apeptide or peptidomimetic that selectively homes to vasculature ofpremalignant pancreas, the peptide or peptidomimetic containing at least5 contiguous amino acids of an amino acid sequence selected from CRSRKG(SEQ ID NO:9) and CEYQLDVE (SEQ ID NO:34), or a conservative variant orpeptidomimetic thereof, or (b) a peptide or peptidomimetic thatselectively homes to premalignant and malignant pancreatic vasculature,the peptide or peptidomimetic containing at least 5 contiguous aminoacids of an amino acid sequence selected from CRGRRST (SEQ ID NO:5) andFRVGVADV (SEQ ID NO:27), or a conservative variant or peptidomimeticthereof, thereby directing the moiety to the vasculature of thepancreatic premalignant lesion. In one embodiment, the moiety is atherapeutic moiety, such as an angiogenic inhibitor. In anotherembodiment, the moiety is a diagnostic moiety.

The invention provides a method of imaging pancreatic premalignantlesions in an individual. The method involves: (a) administering to theindividual a conjugate containing a detectable moiety linked to apeptide or peptidomimetic that selectively homes to vasculature ofpremalignant pancreas, the peptide or peptidomimetic containing at least5 contiguous amino acids of an amino acid sequence selected from CRSRKG(SEQ ID NO:9) and CEYQLDVE (SEQ ID NO:34), or a conservative variant orpeptidomimetic thereof, and (b) detecting the conjugate, thereby imagingpancreatic premalignant lesions.

Further provided by the invention is a method of treating a pancreaticpremalignant lesion in an individual. The method involves administeringto the individual a conjugate containing a therapeutic moiety linked to:(a) a peptide or peptidomimetic that selectively homes to premalignantand malignant pancreatic vasculature, the peptide or peptidomimeticcontaining at least 5 contiguous amino acids of an amino acid sequenceselected from CRGRRST (SEQ ID NO:5), and FRVGVADV (SEQ ID NO:27), or aconservative variant or peptidomimetic thereof, or (b) a peptide orpeptidomimetic that selectively homes to vasculature of premalignantpancreas containing at least 5 contiguous amino acids of an amino acidsequence selected from CRSRKG (SEQ ID NO:9) and CEYQLDVE (SEQ ID NO:34),thereby directing the therapeutic moiety to the pancreatic premalignantlesion in the individual to treat the pancreatic premalignant lesion. Inone embodiment, the moiety is a therapeutic moiety, such as anangiogenic inhibitor.

The invention provides of directing a moiety to pancreatic tumor cellsand pancreatic tumor vasculature in an individual. The method involvesadministering to the individual a conjugate containing a moiety linkedto: (a) a peptide or peptidomimetic that selectively homes to pancreatictumor cells and pancreatic tumor vasculature, the peptide orpeptidomimetic containing at least 5 contiguous amino acids of an aminoacid sequence selected from CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ IDNO:19), FRVGVADV and VGVGEWSV (SEQ ID NO:35), or a conservative variantor peptidomimetic thereof, or (b) a peptide or peptidomimetic thatselectively homes to premalignant and malignant pancreatic vasculature,the peptide or peptidomimetic containing at least 5 contiguous aminoacids of an amino acid sequence selected from CRGRRST (SEQ ID NO:5) andFRVGVADV (SEQ ID NO:27), or a conservative variant or peptidomimeticthereof, thereby directing the moiety to pancreatic tumor cells andpancreatic tumor vasculature.

The invention provides a method of imaging pancreatic tumors andpancreatic tumor vasculature in an individual. The method involves (a)administering to the individual a conjugate containing a detectablemoiety linked to a peptide or peptidomimetic that selectively homes topancreatic tumor cells and pancreatic tumor vasculature, the peptide orpeptidomimetic containing at least 5 contiguous amino acids of an aminoacid sequence selected from CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ IDNO:19) and VGVGEWSV (SEQ ID NO:35), or a conservative variant orpeptidomimetic thereof, and (b) detecting the conjugate, thereby imagingthe pancreatic tumors and pancreatic tumor vasculature.

The invention provides a method of reducing the severity of pancreaticcancer in an individual. The method involves administering to theindividual a conjugate containing a therapeutic moiety linked to:

(a) a peptide or peptidomimetic that selectively homes to pancreatictumor cells and pancreatic tumor vasculature, the peptide orpeptidomimetic containing at least 5 contiguous amino acids of an aminoacid sequence selected from CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ IDNO:19), FRVGVADV and VGVGEWSV (SEQ ID NO:35), or a conservative variantor peptidomimetic thereof, or (b) a peptide or peptidomimetic thatselectively homes to premalignant and malignant pancreatic vasculature,the peptide or peptidomimetic containing at least 5 contiguous aminoacids of an amino acid sequence selected from CRGRRST (SEQ ID NO:5) andFRVGVADV (SEQ ID NO:27), or a conservative variant or peptidomimeticthereof, thereby directing the therapeutic moiety to pancreatic tumorcells or pancreatic tumor vasculature in the individual to reduce theseverity of the pancreatic cancer.

The invention provides a method of staging tumor progression in anindividual having or suspected of having a pancreatic premalignantlesion or pancreatic tumor. The method involves: (a) administering tothe individual at least one conjugate containing a detectable moietylinked to (i) a peptide or peptidomimetic that selectively homes tovasculature of premalignant pancreas, the peptide or peptidomimeticspecifically binding a cognate receptor for CRSRKG (SEQ ID NO:9) orCEYQLDVE (SEQ ID NO:34), or (ii) a peptide or peptidomimetic thatselectively homes to pancreatic tumor cells and pancreatic tumorvasculature, the peptide or peptidomimetic specifically binding acognate receptor for an amino acid sequence selected from CKAAKNK (SEQID NO:15), CKGAKAR (SEQ ID NO:19) and VGVGEWSV (SEQ ID NO:35); and (b)detecting the conjugate, wherein detection of the conjugate containing apeptide or peptidomimetic that selectively homes to vasculature ofpremalignant pancreas indicates a premalignant stage of tumorprogression in the individual and wherein detection of the conjugatecontaining a peptide or peptidomimetic that selectively homes topancreatic tumor cells and pancreatic tumor vasculature indicates amalignant stage of tumor progression in the individual.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that phage-displayed peptides selectively home topremalignant pancreas or pancreatic tumor cells. (A) Ex vivo screeningon angiogenic islets or tumors derived from RIP1-Tag2 mice using theCX₇C peptide library displayed on T7 phage. The enriched phage poolswere used for subsequent in vivo homing in RIP1-Tag2 mice; (B)angiogenic islets (3 rounds of selection); (C) tumors (2 rounds ofselection); (D) In vivo homing of individual phage to RIP1-Tag2angiogenic islets and tumors.

FIG. 2 shows tumor progression stage-specific homing offluorescein-conjugated peptides in RIP1-Tag2 model. Visualization of anangiogenic islet-selective peptide (RSR) homing is shown in normal islet(A), angiogenic islet (B), and tumor (C). Homing profiles are also shownfor a tumor-selective peptide (KAA) to normal islet (D), angiogenicislet (E), and tumor (F), as well as of a peptide (RGR) that homes toboth angiogenic islets and tumors (G) normal islet (H) angiogenic islet,and (I) tumor. Control tissues were (J) kidney, (K) brain, and (L)liver. Magnification shown is 200×.

FIG. 3 shows co-localization of fluorescein-conjugated peptides withvascular markers in RIP1-Tag2 islet lesions. RSR peptide localization inan angiogenic islet is shown in panel A and D, while co-staining forMECA-32 and the merge are shown in panels B and C. Co-staining for NG2is shown in panel E, with the merge in panel F. KAA peptide localizationin a tumor is shown in panels G and J, while co-staining for MECA-32 andthe merge are shown in panels H and I. Co-staining for NG2 is shown inpanel K, with the merge in panel L. RGR peptide localization in anangiogenic islet is shown in panel M and P, while co-staining forMECA-32 and the merge are shown in panels N and O. Co-staining for NG2is shown in panel Q, with the merge in panel R. Scale bar shown is 10mm.

FIG. 4 shows evaluation of the specificity of selected homing phage andpeptides. (A) Bar graph showing homing efficiency of individual phage toa pancreatic islet tumor in a RIP1-Tag2 mouse, a bTC3-derivedsubcutaneous transplant tumor in a nude mouse, and a squamous cellcarcinoma in a K14-HPV16 mouse. (B) Table summarizing the relativehoming of fluorescein-conjugated peptides to different tumor models; +++indicates strong homing, as revealed by the fluorescent intensity ofi.v. injected peptide, ++ indicates moderate homing, + indicates weakhoming, − indicates absence of homing. Representative images offluorescein-conjugated KAR peptide homing to a RIP1-Tag2 pancreaticislet tumor (C), a bTC3 subcutaneous tumor (D), and an MDA subcutaneoustumor (E) are also shown. Magnification shown is 200×.

FIG. 5 shows binding of RGR phage to PDGFRβ. (A) Bar graph showingbinding of RGR or RSR phage to 293 cells transfected with either thePDGFRβ, VEGFR2, or non-transfected cells. (B) Co-localization offluorescein-conjugated RGR-peptide (a) with the PDGFRβ antibody (b) andmerged images (c, d) in RIP1-Tag2. Magnification shown is 400×.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the discovery of homing moleculesthat selectively home to vasculature of premalignant tissues and furtherdirected to molecules that selectively home to tumor cells and tumorvasculature, such as premalignant pancreatic tissue and pancreatic tumorcells and tumor vasculature. As disclosed herein in Example I, peptidesspecific for premalignant pancreatic lesions or for pancreatic tumorcells and pancreatic tumor vasculature were isolated using a combinationof in vivo and ex vivo selections using 12 week old RIP1-Tag2 mice.

The RIP1-Tag2 transgenic mouse is a prototypical mouse model ofmultistage tumorigenesis of islet cell carcinoma (Hanahan, Nature315:115-122 (1985)). RIP1-Tag2 transgenic mice express the SV40 Tantigens (Tag) under the control of the insulin gene promoter, whichelicits the sequential development of tumors in the islets of Langerhansover a period of 12-14 weeks. Hyperplastic islets begin to appear ataround 4 weeks of age, and angiogenesis is activated a few weeks laterin a subset of the hyperplastic islets, producing angiogenic(dysplastic) islets (Bergers et al., Int. J. Dev. Biol. 42:995-1002(1998); Folkman et al., Nature 339:58-61 (1989)). Solid tumors formbeginning at 9-10 weeks, initially presenting as small nodules that growand progress to large islet tumors with well defined margins as well astwo classes of invasive carcinoma (Lopez and Hanahan, Cell 1:339-353(2002)). As is described in Examples I and II, stage-specific molecularmarkers accessible via the circulation were identified, either on thesurface of endothelial cells, their peri-endothelial support cells(pericytes and smooth muscle cells) or even tumor cells themselves (as aresult of the hemorrhagic, leaky angiogenic vasculature). Phage poolsthat homed preferentially to different stages during RIP1-Tag2tumorigenesis were identified using ex vivo and in vitro selections, asdescribed in Examples I and II. Also identified were ‘pan-angiogenic’markers shared by many types of tumors (Example IV).

Ex vivo selections were performed using suspensions of either angiogenicislets or solid tumors, and resulted in a 7 or 8-fold enrichment ofphage relative to enrichment of nonrecombinant phage lacking displayedpeptides, respectively (FIG. 1A). Sequential in vivo selection usingangiogenic islets resulted in a 7-fold enrichment (FIG. 1B); andsequential in vivo using solid tumors resulted in an 8-fold enrichment(FIG. 1C). Peptides displayed on several of the selected phage cloneswere found to be highly selective for either angiogenic islets incomparison to solid tumors, or for solid tumors in comparison toangiogenic islets (FIG. 1D).

Sequencing of phage from the selected pools identified a number ofpeptide sequences that were represented more than once, and these weretested for their ability to bind cell suspensions prepared fromangiogenic islets and tumors. Six of the phage selected for furtheranalysis were from the tumor screen (referred to as KAA, RGR, RSR, VGVA,VGVG and KAR), and one (EYQ) was picked from the angiogenic screen.Peptide sequences corresponding to each of these peptide motifs are setforth as SEQ ID NO:15 (CKAAKNK), SEQ ID NO:5 (CRGRRST), SEQ ID NO:9(CRSRKG), SEQ ID NO:27 (FRVGVADV), SEQ ID NO:35 (VGVGEWSV), SEQ ID NO:19(CKGAKAR) and SEQ ID NO:34 (CEYQLDVE), respectively.

Each of the identified peptides was linear, although the phage libraryused (CX₇C) was designed to express primarily cyclized peptides, with aminority of linear peptides. However, linear peptides form in thislibrary, for example, by the occurrence of a stop codon in a randominsert, causing truncation of the peptide, and by occurrence of aframeshift mutation that mutates the second cysteine (which is requiredfor cyclization) into valine.

To confirm homing specificity of the selected peptides, purifiedpeptides were intravenously injected into 8-week or 12-week old RIP-Tag2mice to examine peptide localization at early (angiogenic) or late(malignant) stages of tumor progression, respectively. The observedpeptide localization profiles in each case was similar to those ofcognate phage. As is described in Example IV, the peptides were found tobe highly selective for premalignant angiogenic islets (RSR and EYQpeptides), malignant solid tumors (KAA and KAR peptides), or bothangiogenic islets and tumors (RGR and VGVA peptides), and did notappreciably home to normal islets, kidney, brain, liver, lung or spleen.

To further characterize homing selectivity, tissues were collectedfollowing i.v. infusion with fluorescein-conjugated RSR, KAA, RGRpeptides, sectioned, and evaluated with endothelial cell and endotheliallumen markers and a neovascular pericyte marker. All three peptides(RSR, KAA, and RGR) co-localized both with the endothelial cell andpericyte markers, indicating that each homes to and binds a moietyassociated with both cell types (see, for example, FIG. 3).Co-localization of these peptides with the same markers in adjacentexocrine pancreas or in normal pancreatic islets was not observed.

As further disclosed in Example IV, homing selectivity of premalignantand/or malignant tissue peptides RGR, KAA, RSR, VGVA, VGVG, and KAR wasanalyzed for the ability of these peptides to home to endothelium intumors of different tissue origins and localized to different anatomicallocations. In particular, two subcutaneously implanted tumors and atumor produced in a transgenic animal model were examined foraccumulation of fluorescein-labeled peptides following intravenousinjection. As shown in FIG. 4A, different homing specificities wereobserved for each peptide in the various tumor microenvironments. Inparticular, as shown in FIG. 4A, peptides KAA, RGR, RSR, VGVA, and VGVGdid not home appreciably to HPV tumor, whereas peptide KAR homed to thistumor type. In addition, peptides KAA, RGR, VGVA, and KAR did not homeappreciably to TC3 tumor, whereas peptides RSR and VGVG homed to thistumor type. As shown in FIG. 4B, peptides RGR and RSR did not homeappreciably to MDA tumor, whereas peptide KAA homed to this tumor type.

As further disclosed in Example V, a receptor for the peptide RGR wasidentified using a combination of sequence searching and cell biologicalmethods. Specifically, using sequence searching methods, a ligand forPDGFR-β was identified to contain the sequence RGRRS. Using cellbiological methods, phage containing the RGR sequence (CRGRRST; SEQ IDNO:5) was shown to bind to PDGFR-β expressing cells, but not to cellsexpressing a similar receptor (VSGFR2) or to control cells. Theassociation of the CRGRRST peptide with PDGFRβ was confirmed whenintravenously injected fluorescein-conjugated CRGRRST peptide was shownto co-localize with PDGFRβ, as visualized by subsequent immunostainingof tissue sections from RIP1-Tag2 tumors.

Peptides and Peptidomimetics

Based on the above findings, the present invention provides severalisolated peptides and peptidomimetics that home to vasculature ofpremalignant pancreas, to pancreatic tumor cells and pancreatic tumorvasculature or to both premalignant and/or malignant pancreaticvasculature. In an embodiment, the invention provides an isolatedpeptide or peptidomimetic that selectively homes to vasculature ofpremalignant pancreas. The peptide contains at least 4 contiguous aminoacids of the amino acid sequence CRSRKG (SEQ ID NO:9) or CEYQLDVE (SEQID NO:34), or a conservative variant or peptidomimetic thereof and has alength of less than 50 residues, such as less than 40 residues, lessthan 30 residues, less than 20 residues, less than 10 residues, or lessthan 8 residues.

In one embodiment, a peptide of the invention, as well as a peptidecontained in a conjugate of the invention, contains at least 4contiguous amino acids of the amino acid sequence CRSRKG (SEQ ID NO:9),or a conservative variant of peptidomimetic thereof. The peptide alsocan contain at least 5 contiguous amino acids of the amino acid sequenceCRSRKG (SEQ ID NO:9), or at least 6 contiguous amino acids of the aminoacid sequence CRSRKG (SEQ ID NO:9), or a conservative variant ofpeptidomimetic thereof. Exemplary peptide sequences that contain atleast 4 contiguous amino acids of the amino acid sequence CRSRKG (SEQ IDNO:9) include, but are not limited to, RSRX₁G wherein X₁ is a basicamino acid (SEQ ID NO:6), CRSRX₁G wherein X₁ is a basic amino acid (SEQID NO:7), and RSRKG (SEQ ID NO:8). X₁ can be, for example, arginine,histidine and lysine.

In another embodiment, a peptide of the invention, as well as a peptidecontained in a conjugate of the invention, contains at least 4contiguous amino acids of the amino acid sequence CEYQLDVE (SEQ IDNO:34), or a conservative variant of peptidomimetic thereof. The peptidealso can contain at least 5 contiguous amino acids of the amino acidsequence CEYQLDVE (SEQ ID NO:34), at least 6 contiguous amino acids ofthe amino acid sequence CEYQLDVE (SEQ ID NO:34), at least 7 contiguousamino acids of the amino acid sequence CEYQLDVE (SEQ ID NO:34), or atleast 8 contiguous amino acids of the amino acid sequence CEYQLDVE (SEQID NO:34) or a conservative variant of peptidomimetic thereof. Exemplarypeptide sequences that contain at least 4 contiguous amino acids of theamino acid sequence CEYQLDVE (SEQ ID NO:34) include, but are not limitedto CEYQL (SEQ ID NO:28), EYQLD (SEQ ID NO:29), EYQLDV (SEQ ID NO:30),EYQLDVE (SEQ ID NO:31), YQLDV (SEQ ID NO:32), and YQLDVE (SEQ ID NO:33).

The invention also provides an isolated peptide or peptidomimetic thatselectively homes to pancreatic tumor cells and pancreatic tumorvasculature. The peptide contains at least 4 contiguous amino acids ofthe amino acid sequence CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ ID NO:19)or VGVGEWSV (SEQ ID NO:35), or a conservative variant or peptidomimeticthereof and has a length of less than 50 residues, such as less than 40residues, less than 30 residues, less than 20 residues, less than 10residues, and less than 8 residues.

In one embodiment, a peptide of the invention, as well as a peptidecontained in a conjugate of the invention, contains at least 4contiguous amino acids of the amino acid sequence CKAAKNK (SEQ IDNO:15), or a conservative variant of peptidomimetic thereof. The peptidealso can contain at least 5 contiguous amino acids of the amino acidsequence CKAAKNK (SEQ ID NO:15), at least 6 contiguous amino acids ofthe amino acid sequence CKAAKNK (SEQ ID NO:15), at least 7 contiguousamino acids of the amino acid sequence CKAAKNK (SEQ ID NO:15), or aconservative variant of peptidomimetic thereof. Exemplary peptidesequences that contain at least 4 contiguous amino acids of the aminoacid sequence CKAAKNK (SEQ ID NO:15) include, but are not limited toCKAX₁K wherein X₁ is a basic amino acid (SEQ ID NO:10), CKAX₁KN whereinX₁ is a basic amino acid (SEQ ID NO:11), CKAAK (SEQ ID NO:12), CKAAKN(SEQ ID NO:13) and KAAKN (SEQ ID NO:14). X₁ can be, for example,arginine, histidine and lysine.

In another embodiment, a peptide of the invention, as well as a peptidecontained in a conjugate of the invention, contains at least 4contiguous amino acids of the amino acid CKGAKAR (SEQ ID NO:19), or aconservative variant of peptidomimetic thereof. The peptide also cancontain at least 5 contiguous amino acids of the amino acid sequenceCKGAKAR (SEQ ID NO:19), at least 6 contiguous amino acids of the aminoacid sequence CKGAKAR (SEQ ID NO:19), at least 7 contiguous amino acidsof the amino acid sequence CKGAKAR (SEQ ID NO:19) or a conservativevariant of peptidomimetic thereof. Exemplary peptide sequences thatcontain at least 4 contiguous amino acids of the amino acid sequenceCKGAKAR (SEQ ID NO:19) include, but are not limited to AKAR (SEQ IDNO:16), GAKAR (SEQ ID NO:17), KGAKAR (SEQ ID NO:18), and CKGAKA (SEQ IDNO:20).

In a further embodiment, a peptide of the invention, as well as apeptide contained in a conjugate of the invention, contains at least 4contiguous amino acids of the amino acid sequence VGVGEWSV (SEQ IDNO:35), or a conservative variant of peptidomimetic thereof. The peptidealso can contain at least 5 contiguous amino acids of the amino acidsequence VGVGEWSV (SEQ ID NO:35), at least 6 contiguous amino acids ofthe amino acid sequence VGVGEWSV (SEQ ID NO:35), at least 7 contiguousamino acids of the amino acid sequence VGVGEWSV (SEQ ID NO:35), or atleast 8 contiguous amino acids of the amino acid sequence VGVGEWSV (SEQID NO:35), or a conservative variant of peptidomimetic thereof.Exemplary peptide sequences that contain at least 4 contiguous aminoacids of the amino acid sequence VGVGEWSV (SEQ ID NO:35) include, butare not limited to VGVG (SEQ ID NO:36), VGVGE (SEQ ID NO:37).

The invention also provides an isolated peptide or peptidomimetic thatselectively homes to premalignant and malignant pancreatic vasculature.The peptide contains at least 4 contiguous amino acids of the amino acidsequence CRGRRST (SEQ ID NO:5) or FRVGVADV (SEQ ID NO:27), or aconservative variant or peptidomimetic thereof and has a length of lessthan 50 residues, such as less than 40 residues, less than 30 residues,less than 20 residues, less than 10 residues, and less than 8 residues.

In one embodiment, a peptide of the invention, as well as a peptidecontained in a conjugate of the invention, contains at least 4contiguous amino acids of the amino acid sequence CRGRRST (SEQ ID NO:5),or a conservative variant of peptidomimetic thereof. The peptide alsocan contain at least 5 contiguous amino acids of the amino acid sequenceCRGRRST (SEQ ID NO:5), at least 6 contiguous amino acids of the aminoacid sequence CRGRRST (SEQ ID NO:5), or at least 7 contiguous aminoacids of the amino acid sequence CRGRRST (SEQ ID NO:5), or aconservative variant of peptidomimetic thereof. Exemplary peptidesequences that contain at least 4 contiguous amino acids of the aminoacid sequence CRGRRST (SEQ ID NO:5) include, but are not limited to,RGRR (SEQ ID NO:1); RGRRS (SEQ ID NO:2); RGRRST (SEQ ID NO:3); andCRGRRS (SEQ ID NO:4).

In another embodiment, a peptide of the invention, as well as a peptidecontained in a conjugate of the invention, contains at least 4contiguous amino acids of the amino acid sequence FRVGVADV (SEQ IDNO:27), or a conservative variant of peptidomimetic thereof. The peptidealso can contain at least 5 contiguous amino acids of the amino acidsequence FRVGVADV (SEQ ID NO:27), at least 6 contiguous amino acids ofthe amino acid sequence FRVGVADV (SEQ ID NO:27), at least 7 contiguousamino acids of the amino acid sequence FRVGVADV (SEQ ID NO:27), or atleast 8 contiguous amino acids of the amino acid sequence FRVGVADV (SEQID NO:27). Exemplary peptide sequences that contain at least 4contiguous amino acids of the amino acid sequence FRVGVADV (SEQ IDNO:27) include, but are not limited to, RVGV (SEQ ID NO:21), RVGVA (SEQID NO:22), RVGVAD (SEQ ID NO:23), VGVAD (SEQ ID NO:24), VGVADV (SEQ IDNO:25), and RVGVADV (SEQ ID NO:26).

As disclosed herein in Example V, a receptor for peptide sequenceCRGRRST (SEQ ID NO:5) was identified as PDGFRβ. Therefore, the inventionprovides an isolated peptide or peptidomimetic that selectively homes topremalignant and malignant pancreatic vasculature, wherein the peptideor peptidomimetic has an ability to bind to a PDGFRβ, and has a lengthof less than 50 residues. The isolated peptide or peptidomimetic canhave, for example, a length of less than 40 residues, less than 30residues, less than 20 residues, less than 10 residues, and less than 8residues.

It is understood that a homing molecule useful in the invention can be,without limitation, a peptide or peptidomimetic. As used herein, theterm “peptide” is used broadly to mean peptides, proteins, fragments ofproteins and the like. The term “peptidomimetic,” as used herein, meansa peptide-like molecule that has the activity of the peptide upon whichit is structurally based. Such peptidomimetics include chemicallymodified peptides, peptide-like molecules containing non-naturallyoccurring amino acids, and peptoids, and have an activity such as theselective homing activity of the peptide upon which the peptidomimeticis derived (see, for example, Goodman and Ro, Peptidomimetics for DrugDesign, in “Burger's Medicinal Chemistry and Drug Discovery” Vol. 1 (ed.M. E. Wolff; John Wiley & Sons (1995), pages 803-861).

A variety of peptidomimetics are known in the art including, but notlimited to, peptide-like molecules that contain a constrained aminoacid, a non-peptide component that mimics peptide secondary structure,or an amide bond isostere. A peptidomimetic that contains a constrained,non-naturally occurring amino acid can include, without limitation, anα-methylated amino acid; α, α-dialkylglycine or α-aminocycloalkanecarboxylic acid; an N^(α)—C^(α) cyclized amino acid; an N^(α)-methylatedamino acid; a β- or γ-amino cycloalkane carboxylic acid; anα,β-unsaturated amino acid; a β,β-dimethyl or β-methyl amino acid; aβ-substituted-2,3-methano amino acid; an N—C^(δ) or C^(α)—C^(δ) cyclizedamino acid; a substituted proline or another amino acid mimetic. Apeptidomimetic that mimics peptide secondary structure can contain,without limitation, a nonpeptidic β-turn mimic; γ-turn mimic; mimic ofβ-sheet structure; or mimic of helical structure, each of which is wellknown in the art. As non-limiting examples, a peptidomimetic also can bea peptide-like molecule that contains an amide bond isostere such as aretro-inverso modification; reduced amide bond; methylenethioether ormethylene-sulfoxide bond; methylene ether bond; ethylene bond; thioamidebond; trans-olefin or fluoroolefin bond; 1,5-disubstituted tetrazolering; ketomethylene or fluoroketomethylene bond or another amideisostere. One skilled in the art understands that these and otherpeptidomimetics are encompassed within the meaning of the term“peptidomimetic” as used herein.

Methods for identifying a peptidomimetic are well known in the art andinclude, for example, the screening of databases that contain librariesof potential peptidomimetics. For example, the Cambridge StructuralDatabase contains a collection of greater than 300,000 compounds thathave known crystal structures (Allen et al., Acta Crystallogr. SectionB, 35:2331 (1979)). This structural depository is continually updated asnew crystal structures are determined and can be screened for compoundshaving suitable shapes, for example, the same shape as a peptide of theinvention, as well as potential geometrical and chemical complementarityto a target molecule. Where no crystal structure of a peptide of theinvention is available, a structure can be generated using, for example,the program CONCORD (Rusinko et al., J. Chem. Inf. Comput. Sci. 29:251(1989)). Another database, the Available Chemicals Directory (MolecularDesign Limited, Informations Systems; San Leandro, Calif.), containsabout 100,000 compounds that are commercially available and also can besearched to identify potential peptidomimetics of a peptide of theinvention, for example, with activity in selectively homing tovasculature of premalignant pancreas or pancreatic tumor cells andpancreatic tumor vasculature.

The peptides and peptidomimetics of the invention, including thebifunctional and multivalent peptides and peptidomimetics describedherein below, can have a variety of lengths. A peptide or peptidomimeticof the invention, or the peptide or peptidomimetic portion of aconjugate of the invention, can have, for example, a relatively shortlength of less than 6, less than 7, less than 8, less than 9, less than10, less than 12, less than 15, less than 20, less than 25, less than30, less than 35, less than 40, less than 45, less than 50, less than60, less than 70 or less than 80 residues. A peptide or peptidomimeticof the invention, or conjugate containing the peptide or peptidomimetic,also can be useful in the context of a significantly longer sequence asdescribed further below. As used herein, the term “residue” refers to anamino acid or analog thereof.

In various embodiments, the peptide or peptidomimetic portion of theconjugate has a defined length. The peptide or peptidomimetic, or thepeptide or peptidomimetic portion of the conjugate, can have, forexample, a length of at most 10, at most 20, most 10, at most 30, most10, at most 40, most 10, at most 50, most 10, at most 100, most 10, atmost 150, most 10, at most 200, most 10, at most 250, most 10, at most300, most 10, at most 400, most 10, at most 500, most 10, at most 600,most 10, at most 700, most 10, at most 800, most 10, at most 900, most10, at most 1000 or most 10, at most 2000 residues. The peptide orpeptidomimetic, or the peptide or peptidomimetic portion of theconjugate, also can have, for example, a length of less than 60, lessthan 50, less than 40, less than 30, less than 25, less than 20, lessthan 15, or less than 10 residues. It is understood that the term“peptide or peptidomimetic portion of the conjugate” means the totalnumber of residues in the peptide or peptidomimetic and any contiguousprotein, peptide or peptidomimetic, such as a therapeutic protein orpro-apoptotic peptide.

Chimeric Proteins

As disclosed herein, a peptide or peptidomimetic of the invention canmaintain homing activity in the context of a significantly longersequence. As a non-limiting example, the peptides referenced as SEQ IDNOS:5, 9, 15, 19, 27, 34 and 35 demonstrated homing to respectivetargets when fused to a phage coat protein, confirming that a peptide ofthe invention can have selective homing activity when embedded in alarger protein sequence (see Example I). Thus, the invention provides achimeric protein containing a peptide or peptidomimetic of theinvention, fused to a heterologous protein. In one embodiment, theinvention provides a chimeric protein containing a peptide orpeptidomimetic that selectively homes to vasculature of premalignantpancreas and contains at least 5 contiguous amino acids of an amino acidsequence selected from SEQ ID NO:9 or SEQ ID NO:34, or a conservativevariant or peptidomimetic of one or these sequences, fused to aheterologous protein.

In another embodiment, the invention provides a chimeric proteincontaining a peptide or peptidomimetic that selectively homes topancreatic tumor cells and pancreatic tumor vasculature and contains atleast 5 contiguous amino acids of an amino acid sequence selected fromSEQ ID NO:15, SEQ ID NO:19, or SEQ ID NO:35, or a conservative variantor peptidomimetic of one or these sequences, fused to a heterologousprotein.

In a further embodiment, the invention provides a chimeric proteincontaining a peptide or peptidomimetic that selectively homes topremalignant and malignant pancreatic vasculature and contains at least5 contiguous amino acids of an amino acid sequence selected from SEQ IDNO:5 or SEQ ID NO:27, or a conservative variant or peptidomimetic of oneor these sequences, fused to a heterologous protein.

A variety of heterologous proteins can be fused to one of these peptidesor peptidomimetics. The term “heterologous,” as used herein in referenceto a protein fused to a peptide or peptidomimetic of the invention,means a protein derived from a source other than the gene encoding thefused peptide or upon which the fused homing peptidomimetic is derived.A heterologous protein can be, without limitation, a heterologousprotein having a therapeutic activity, an antibody or an antigen-bindingfragment. A chimeric protein of the invention can have a variety oflengths including, but not limited to, up to 100, up to 200, up to 300,up to 400, up to 500, up to 800, up to 1000 or up to 2000 residues ormore.

Bifunctional Peptides

The invention also provides bifunctional peptides that contain a peptidethat selectively homes to vasculature of premalignant pancreas fused toa second peptide having a separate function. Also provided by theinvention are bifunctional peptides that contain a peptide thatselectively homes pancreatic tumor cells and pancreatic tumorvasculature fused to a second peptide having a separate function.Further provided by the invention are bifunctional peptides that containa peptide that selectively homes to premalignant or malignant pancreaticvasculature fused to a second peptide having a separate function. Suchbifunctional peptides have at least two functions conferred by differentportions of the peptide and can, for example, display anti-angiogenicactivity or pro-apoptotic activity in addition to selective homingactivity. As non-limiting examples, the invention provides bifunctionalpeptides such as CRSRKG-GG-_(D)(KLAKLAK)₂, CEYQLDVE-GG-_(D)(KLAKLAK)₂,CRGRRST-GG-_(D)(KLAKLAK)₂, CKAAKNK-GG-_(D)(KLAKLAK)₂,FRVGVADV-GG-_(D)(KLAKLAK)₂, CSRPPRSEC-GG-_(D)(KLAKLAK)₂,VGVGEWSV-GG-_(D)(KLAKLAK)₂ or CKGAKAR-GG-_(D)(KLAKLAK)₂. In suchpeptides, the CRSRKG (SEQ ID NO:9), CEYQLDVE (SEQ ID NO:34), CRGRRST(SEQ ID NO:5), CKAAKNK (SEQ ID NO:15), FRVGVADV (SEQ ID NO:27), VGVGEWSV(SEQ ID NO:35), or CKGAKAR (SEQ ID NO:19) portion exhibits selectivehoming activity, while the _(D)(KLAKLAK)₂ portion exhibits pro-apoptoticactivity.

Homing Molecules that are Antibodies

The conjugates and methods of the invention can be practiced with ahoming antibody or antigen-binding fragment thereof that selectivelyhomes to vasculature of premalignant pancreas; that selectively homes topancreatic tumor cells and pancreatic tumor vasculature or thatselectively homes to premalignant and malignant pancreatic vasculature.As used herein, the term “antibody” is used in its broadest sense toinclude polyclonal and monoclonal antibodies, as well as polypeptidefragments of antibodies that retain binding activity for the respectivecognate receptor of at least about 1×10⁵ M⁻¹. One skilled in the artunderstands that antibody fragments including, without limitation, Fab,F(ab′)₂ and Fv fragments, can retain binding activity for a cognatereceptor and, thus, are included within the definition of antibody. Inaddition, the term “antibody,” as used herein, encompasses non-naturallyoccurring antibodies and fragments usually containing, at a minimum, oneV_(H) and one V_(L) domain, such as chimeric antibodies, humanizedantibodies and single chain Fv fragments (scFv) that specifically orselectively bind the appropriate cognate receptor. Such non-naturallyoccurring antibodies can be constructed using solid phase peptidesynthesis, produced recombinantly or obtained by screeningphage-displayed or other combinatorial libraries such as thoseconsisting of variable heavy and light chains as described in Borrebaeck(Ed.), Antibody Engineering (Second edition) New York: Oxford UniversityPress (1995)) using, for example, an assay described herein below.

Homing molecules that are antibodies also can be prepared using acognate receptor fusion protein or a synthetic peptide encoding aportion of a cognate receptor. One skilled in the art understands thatpurified human or other cognate receptors, which can be producedrecombinantly, including peptide portions of a cognate receptor such assynthetic peptide fragments can be used as immunogens. It is understoodthat fragments of the cognate receptor for an amino acid sequenceselected from SEQ ID NOS:5, 9, 15, 19, 27, 34 or 35 useful as immunogensinclude fragments of the cognate receptor that serve to produceanti-cognate receptor antibodies that are readily internalized intocells expressing cell-surface cognate receptor for an amino acidsequence selected from SEQ ID NOS:5, 9, 15, 19, 27, 34 or 35. Oneskilled in the art further understands that non-immunogenic fragments orsynthetic peptides of a cognate receptor for an amino acid sequenceselected from SEQ ID NOS:5, 9, 15, 19, 27, 34 or 35 can be madeimmunogenic by coupling the hapten to a carrier molecule such as bovineserum albumin (BSA) or keyhole limpet hemocyanin (KLH). In addition,various other carrier molecules and methods for coupling a hapten to acarrier molecule are well known in the art as described, for example, byHarlow and Lane, Antibodies: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1988)).

Conjugates

The invention provides a conjugate that includes a therapeutic moietylinked to a peptide or peptidomimetic that selectively homes tovasculature of premalignant pancreas. The peptide or peptidomimeticcontains at least 5 contiguous amino acids of an amino acid sequenceselected from CRSRKG (SEQ ID NO:9) and CEYQLDVE (SEQ ID NO:34), or aconservative variant or peptidomimetic thereof. In embodiments of theinvention, the peptide or peptidomimetic has a length of less than 100residues, less than 50 residues and less than 25 residues. Also providedby the invention is a conjugate containing a therapeutic moiety linkedto a peptide or peptidomimetic that selectively homes to vasculature ofpremalignant pancreas, in which the peptide or peptidomimetic bindsspecifically to a cognate receptor for SEQ ID NO:9 or SEQ ID NO:34.

The invention additionally provides a conjugate containing a therapeuticmoiety linked to a peptide or peptidomimetic that selectively homes topancreatic tumor cells and pancreatic tumor vasculature, the peptide orpeptidomimetic comprising at least 5 contiguous amino acids of an aminoacid sequence selected from CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ IDNO:19), and VGVGEWSV (SEQ ID NO:35), or a conservative variant orpeptidomimetic thereof. In embodiments of the invention, the peptide orpeptidomimetic has a length of less than 100 residues, less than 50residues and less than 25 residues. Also provided by the invention is aconjugate that contains therapeutic moiety linked to a peptide orpeptidomimetic that selectively homes to pancreatic tumor cells andpancreatic tumor vasculature, wherein the peptide or peptidomimeticbinds specifically to a cognate receptor for SEQ ID NO:15, SEQ ID NO:19or SEQ ID NO:35.

The invention provides a conjugate that contains a therapeutic moietylinked to a peptide or peptidomimetic that selectively homes topremalignant and malignant pancreatic vasculature, the peptide orpeptidomimetic containing at least 5 contiguous amino acids of an aminoacid sequence selected from CRGRRST (SEQ ID NO:5) and FRVGVADV (SEQ IDNO:27), or a conservative variant or peptidomimetic thereof. Inembodiments of the invention, the peptide or peptidomimetic has a lengthof less than 100 residues, less than 50 residues and less than 25residues. Also provided by the invention is a conjugate that contains atherapeutic moiety linked to a peptide or peptidomimetic thatselectively homes to pancreatic tumor cells and pancreatic tumorvasculature, wherein the peptide or peptidomimetic binds specifically toa cognate receptor for SEQ ID NO:5 or SEQ ID NO:27.

The conjugates and methods of the invention disclosed herein involvehoming molecules. As used herein, the term “molecule” is used broadly tomean a polymeric or non-polymeric organic chemical such as a smallmolecule drug; a nucleic acid molecule such as an RNA, a cDNA or otherDNA, or an oligonucleotide; a peptide or peptidomimetic; or a proteinsuch as an antibody or a growth factor receptor or a fragment thereofsuch as an Fv, Fd, or Fab fragment of an antibody containing theantigen-binding domain.

The phrase “homing molecule that selectively homes to vasculature ofpremalignant pancreas,” as used herein, means any molecule thatpreferentially localizes in vivo to vasculature of premalignant pancreasas compared to vasculature of malignant pancreas and vasculature ofnormal pancreas. Similarly, the phrase “peptide or peptidomimetic thatselectively homes to vasculature of premalignant pancreas” means apeptide or peptidomimetic that preferentially localizes in vivo tovasculature of premalignant pancreas as compared to vasculature ofmalignant pancreas and vasculature of normal pancreas. As disclosedherein, such a homing molecule can be a peptide or peptidomimetic. It isunderstood that a peptide or peptidomimetic that selectively homes tovasculature of premalignant pancreas can home to the supportingvasculature of a variety of premalignant lesions in addition topremalignant pancreas, or can exhibit preferential homing to vasculatureof premalignant lesions in a subset of tissue types includingpremalignant pancreas, or can exhibit significant homing exclusively tovasculature of premalignant pancreas.

Selective homing of a peptide or peptidomimetic that selectively homesto vasculature of premalignant pancreas generally is characterized by atleast a two-fold greater localization within vasculature of premalignantpancreas as compared to vasculature of malignant pancreas and normalpancreatic vasculature. Such a peptide or peptidomimetic can becharacterized, for example, by 5-fold, 10-fold, 20-fold or more greaterlocalization within vasculature of premalignant pancreas as compared tovasculature of malignant pancreas and normal pancreatic vasculature. Asdiscussed above, it is understood that a peptide or peptidomimetic thatselectively homes to vasculature of premalignant pancreas can home, inpart, to vasculature of one or more other premalignant tissues.

As used herein, the term “premalignant” means a precancerous state of atissue having a an abnormality in which cancer is more likely to occurthan in a normal tissue of the same type. Such an abnormality can becharacterized based on histological abnormalities of cytology and/orarchitecture or biochemical differences between the precancerous versusnormal states of the tissue. Particular differences depend on theparticular type of tissue undergoing a premalignant process and aredescribed in the art, for example, as metaplasia, dysplasia,hyperplasia, carcinoma in situ, angiogenic and the like, depending onthe degree of structural and/or functional change compared to normal.

The phrase “homing molecule that selectively homes to pancreatic tumorcells and pancreatic tumor vasculature,” as used herein, means anymolecule that preferentially localizes in vivo to pancreatic tumor cellsand pancreatic tumor vasculature as compared to premalignant pancreatictumor cells and vasculature, and normal pancreatic tumor cells andvasculature. Similarly, the phrase “peptide or peptidomimetic thatselectively homes to pancreatic tumor cells and pancreatic tumorvasculature” means a peptide or peptidomimetic that preferentiallylocalizes in vivo to pancreatic tumor cells and pancreatic tumorvasculature as compared to premalignant pancreatic cells andvasculature, and normal pancreatic cells and vasculature. It isunderstood that a peptide or peptidomimetic that selectively homes topancreatic tumor cells and pancreatic tumor vasculature can home to thesupporting vasculature of a variety of malignant lesions in addition topancreatic tumor cells and vasculature, or can exhibit preferentialhoming to vasculature of premalignant lesions in a subset of tissuetypes including premalignant pancreas, or can exhibit significant homingexclusively to pancreatic tumor cells and pancreatic tumor vasculature.

Selective homing of a peptide or peptidomimetic that selectively homesto pancreatic tumor cells and pancreatic tumor vasculature generally ischaracterized by at least a two-fold greater localization withinpancreatic tumor cells and pancreatic tumor vasculature as compared topancreatic premalignant cells and premalignant pancreatic vasculatureand normal pancreatic cells and vasculature. Such a peptide orpeptidomimetic can be characterized, for example, by 5-fold, 10-fold,20-fold or more greater localization within pancreatic tumor cells andpancreatic tumor vasculature as compared to premalignant cells andpremalignant pancreatic vasculature and normal pancreatic cells andvasculature. As discussed above, it is understood that a peptide orpeptidomimetic that selectively homes to pancreatic tumor cells andpancreatic tumor vasculature can additionally localize to tumor cellsand vasculature of one or more other malignant tissues in addition toselectively homing to pancreatic tumor cells and pancreatic tumorvasculature.

A peptide or peptidomimetic of the invention can be characterized byhaving the ability to preferentially localize to both premalignant andmalignant pancreatic vasculature. Such a peptide or peptidomimetic canhave greater selectivity for premalignant vasculature, greaterselectivity for malignant vasculature, or can have similar selectivitiesfor both premalignant and malignant vasculature. The phrase “homingmolecule that selectively homes to premalignant and malignant pancreaticvasculature,” as used herein, means any molecule that preferentiallylocalizes in vivo to premalignant and malignant pancreatic vasculatureas compared to normal pancreatic vasculature. Similarly, the phrase“peptide or peptidomimetic that selectively homes to premalignant andmalignant pancreatic vasculature” means a peptide or peptidomimetic thatpreferentially localizes in vivo to premalignant and malignantpancreatic vasculature as compared to normal pancreatic vasculature. Itis understood that a peptide or peptidomimetic that selectively homes topremalignant and malignant pancreatic vasculature can home to thesupporting vasculature of a variety of premalignant and malignantlesions in addition to premalignant and malignant pancreaticvasculature, or can exhibit preferential homing to vasculature ofpremalignant and malignant lesions in a subset of tissue types includingpremalignant and malignant pancreas, or can exhibit significant homingexclusively to premalignant and malignant pancreatic vasculature. It isalso understood that a peptide or peptidomimetic that selectively homesto premalignant and malignant pancreatic vasculature can home to tumorcells, such as pancreatic tumor cells, for example, via compromised,damaged or otherwise leaky malignant pancreatic vasculature.

Selective homing of a peptide or peptidomimetic that selectively homesto premalignant and malignant pancreatic vasculature generally ischaracterized by at least a two-fold greater localization withinpremalignant or malignant pancreatic vasculature as compared tovasculature of normal pancreas. Such a peptide or peptidomimetic can becharacterized, for example, by 5-fold, 10-fold, 20-fold or more greaterlocalization within premalignant and malignant pancreatic vasculature ascompared to vasculature of normal pancreas. As discussed above, it isunderstood that a peptide or peptidomimetic that selectively homes topremalignant and malignant pancreatic vasculature can additionallylocalize to premalignant and malignant vasculature of one or more othertissues in addition to selectively homing to premalignant and malignantpancreatic vasculature. In addition, a peptide or peptidomimetic thatselectively homes to vasculature of premalignant pancreas that alsoselectively homes, to a lesser extent, to pancreatic tumor cells andpancreatic tumor vasculature can be characterized as a peptide orpeptidomimetic that selectively homes to premalignant and malignantpancreatic vasculature. Similarly, a peptide or peptidomimetic thatselectively homes to pancreatic tumor cells and pancreatic tumorvasculature that also selectively homes, to a lesser extent, topremalignant pancreas can be characterized as a peptide orpeptidomimetic that selectively homes to premalignant and malignantpancreatic vasculature.

Conservative Variants

The present invention also provides a peptide or peptidomimetic orconjugate containing a peptide or peptidomimetic that includes an aminoacid sequence that is a conservative variant, for example, of at leastfive contiguous amino acids of an amino acid sequence selected fromCRGRRST (SEQ ID NO:5), CRSRKG (SEQ ID NO:9); CKAAKNK (SEQ ID NO:15),CKGAKAR (SEQ ID NO:19), FRVGVADV (SEQ ID NO:27), CEYQLDVE (SEQ IDNO:34), and VGVGEWSV (SEQ ID NO:35). As used herein, a “conservativevariant” is an amino acid sequence in which a first amino acid isreplaced by a second amino acid or amino acid analog having at least onesimilar biochemical property, which can be, for example, similar size,charge, hydrophobicity or hydrogen-bonding capacity. For example, afirst hydrophobic amino acid can be conservatively substituted with asecond (non-identical) hydrophobic amino acid such as alanine, valine,leucine, or isoleucine, or an analog thereof. Similarly, a first basicamino acid can be conservatively substituted with a second basic aminoacid such as arginine or lysine, or an analog thereof. In the same way,a first acidic amino acid can be conservatively substituted with asecond acidic amino acid such as aspartic acid or glutamic acid, or ananalog thereof, or an aromatic amino acid such as phenylalanine can beconservatively substituted with a second aromatic amino acid or aminoacid analog, for example, tyrosine.

Multivalent Conjugates

The invention provides a multivalent conjugate, containing a therapeuticmoiety linked to at least two peptides or peptidomimetics thatselectively home to vasculature of premalignant pancreas, each of thepeptides or peptidomimetics containing at least 5 contiguous amino acidsof an amino acid sequence selected from CRSRKG (SEQ ID NO:9) andCEYQLDVE (SEQ ID NO:34).

The invention further provides a multivalent conjugate that contains atherapeutic moiety linked to at least two peptides or peptidomimeticsthat selectively home to pancreatic tumor cells and pancreatic tumorvasculature, each of the peptides or peptidomimetics containing at least5 contiguous amino acids of an amino acid sequence selected from CKAAKNK(SEQ ID NO:15), CKGAKAR (SEQ ID NO:19), and VGVGEWSV (SEQ ID NO:35), ora conservative variant or peptidomimetic thereof.

The invention provides a multivalent conjugate that contains atherapeutic moiety linked to at least two peptides or peptidomimeticsthat selectively home to premalignant and malignant vasculature, each ofthe peptides or peptidomimetics containing at least 5 contiguous aminoacids of an amino acid sequence selected from CRGRRST (SEQ ID NO:5) andFRVGVADV (SEQ ID NO:27), or a conservative variant or peptidomimeticthereof.

A multivalent conjugate of the invention containing multiple peptide orpeptidomimetics can include, for example, two or more, three or more,five or more, ten or more, twenty or more, thirty or more, forty ormore, fifty or more, 100 or more, 200 or more, 300 or more, 400 or more,500 or more or 1000 or more peptides or peptidomimetics. In oneembodiment, the peptides or peptidomimetics have an identical amino acidsequence. In another embodiment, the multivalent conjugate includespeptides or peptidomimetics having non-identical amino acid sequences.

A multivalent conjugate of the invention can be linked to a variety ofmoieties. Moieties useful in a multivalent conjugate of the inventioninclude, without limitation, phage, retroviruses, adenoviruses,adeno-associated viruses and other viruses, cells, liposomes, polymericmatrices, non-polymeric matrices or particles such as gold particles,microdevices and nanodevices, and nano-scale semiconductor materials.

Liposomes consisting, without limitation, of phospholipids or otherlipids, are nontoxic, physiologically acceptable and metabolizablecarriers that are readily made to be incorporated into a conjugate ofthe invention (Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, BocaRaton, Fla. (1984)). It is understood that the liposome or otherpolymeric matrix additionally can include one or more other componentsif desired, such as, without limitation, one or any combination oftherapeutic agents, anti-angiogenic agents or cytotoxic agents.

As disclosed herein, peptides CEYQLDVE (SEQ ID NO:34) and CRSRKG (SEQ IDNO:9) recognize a target “receptor” that is expressed in vasculature ofpremalignant pancreas but is essentially absent or inaccessible forbinding via the circulation in vasculature of malignant pancreas ornormal pancreas. Also as disclosed herein, peptides CKAAKNK (SEQ IDNO:15); CKGAKAR (SEQ ID NO:19); and VGVG (SEQ ID NO:x) recognize atarget “receptor” that is expressed in pancreatic tumor cells andpancreatic tumor vasculature but is essentially absent or inaccessiblefor binding via the circulation in the vasculature or premalignantpancreas or normal pancreas. Further, as disclosed herein, peptidesCRGRRST (SEQ ID NO:5) or FRVGVADV (SEQ ID NO:27) recognize a target“receptor” that is expressed in premalignant and malignant pancreaticvasculature but is essentially absent or inaccessible for binding viathe circulation in the vasculature of normal pancreas. The cell surfaceand cell-type selective expression of the target receptor form the basisfor the selective homing activity of the specific peptides describedabove and related peptides, peptidomimetics and other molecules.

Based on these discoveries, it is clear that molecules structurallyunrelated to SEQ ID NOS: 9 or 34 but that bind the same cognatereceptors also have the same characteristic of selectively homing tovasculature of premalignant pancreas and other premalignant tissues.Such molecules can be identified by the ability to specifically bind to,or to compete with SEQ ID NO:9 or 34 for specific binding to cellsexpressing their respective cognate receptors. It is also clear thatmolecules structurally unrelated to SEQ ID NOS:15 and 19 but that bindthe same cognate receptors also have the same characteristic ofselectively homing to pancreatic tumor cells and pancreatic tumorvasculature and cells and vasculature of other malignant tissues. Suchmolecules can be identified by the ability to specifically bind to, orto compete with SEQ ID NOS:15 or 19 for specific binding to cellsexpressing their respective cognate receptors. Further, it is clear thatmolecules structurally unrelated to SEQ ID NOS:5, 27 or 35 but that bindthe same cognate receptors also have the same characteristic ofselectively homing to premalignant and malignant pancreatic vasculatureand premalignant and malignant vasculature of other tissues. Suchmolecules can be identified by the ability to specifically bind to, orto compete with SEQ ID NOS:5, 27 or 35 for specific binding to cellsexpressing their respective cognate receptors. Selective homing tovasculature of premalignant pancreas; pancreatic tumor cells andpancreatic tumor vasculature; or premalignant and malignant pancreaticvasculature, readily can be confirmed using in vivo panning as disclosedherein in Example I (see, also, U.S. Pat. No. 5,622,699).

A homing molecule of the invention, such as a peptide or peptidomimetic,specifically binds the indicated cognate receptor. As used herein, theterm “specifically binds” or “specifically binding” means binding thatis measurably different from a non-specific interaction. Specificbinding can be measured, for example, by determining binding of amolecule compared to binding of a control molecule, which generally is amolecule of similar structure that does not have binding activity. Inthis case, specific binding is indicated if the molecule has measurablyhigher affinity for cells expressing the cognate receptor, for example,than for cells that do not express the cognate receptor. Specificity ofbinding can be determined, for example, by competitive inhibition of thebinding of a known binding molecule such SEQ ID NO:9 or 34 to identifymolecules that selectively home to vasculature of premalignant pancreas;by competitive inhibition of the binding of a known binding moleculesuch as SEQ ID NO:15, 19 or 35 to identify molecules that selectivelyhome to pancreatic tumor cells and pancreatic tumor vasculature; or bycompetitive inhibition of the binding of a known binding molecule suchas SEQ ID NO:5 or 27 to identify molecules that selectively home topremalignant and malignant pancreatic vasculature.

The term “binds specifically,” as used herein, includes both low andhigh affinity specific binding. Specific binding can be exhibited, forexample, by a low affinity homing molecule having a Kd of at least about10⁻⁴ M. For example, if the cognate receptor has more than one bindingsite, a homing molecule having low affinity can be useful for targeting,for example, vasculature of premalignant pancreas; pancreatic tumorcells and pancreatic tumor vasculature; or premalignant and malignantpancreatic vasculature. Specific binding also can be exhibited by a highaffinity homing molecule, for example, a homing molecule having a Kd ofat least about 10⁻⁵ M. Such a molecule can have, for example, a Kd of atleast about 10⁻⁶ M, at least about 10⁻⁷ M, at least about 10⁻⁸ M, atleast about 10⁻⁹ M, at least about 10⁻¹⁰ M, or can have a Kd of at leastabout 10⁻¹¹ M or 10⁻¹² M or greater. Both low and high affinity homingmolecules are useful and are encompassed by the invention. Low affinityhoming molecules are useful in targeting, for example, multivalentconjugates such as viruses and other particles. High affinity homingmolecules are useful in targeting, for example, multivalent andunivalent conjugates.

Thus, the invention further provides a conjugate that contains atherapeutic moiety linked to a peptide or peptidomimetic thatselectively homes to vasculature of premalignant pancreas and thatspecifically binds a cognate receptor for SEQ ID NO:9 or 34. Alsoprovided is a conjugate that contains a therapeutic moiety linked to apeptide or peptidomimetic that selectively homes to pancreatic tumorcells and pancreatic tumor vasculature and which specifically binds acognate receptor for an amino acid sequence selected from SEQ ID NO:15,19 or 35. Further provided is a conjugate that contains a therapeuticmoiety linked to a peptide or peptidomimetic that selectively homes topremalignant and malignant pancreatic vasculature and that specificallybinds a cognate receptor for SEQ ID NO:5 or 27. As is shown herein inExample V, the PDGFRβ is a cognate receptor for SEQ ID NO:5. Thus, inone embodiment, the conjugate contains a peptide or peptidomimetic thatspecifically binds PDGFRβ.

In one embodiment, any of such conjugates can contain a peptide orpeptidomimetic that is not an antibody or antigen-binding fragmentthereof. In another embodiment, the peptide or peptidomimetic portion ofthe conjugate can have a length of at most 200 residues, or a length ofat most 50 residues.

The invention provides method of directing a moiety to a pancreaticpremalignant lesion in an individual. The method involves administeringto the individual a conjugate containing a moiety linked to (a) apeptide or peptidomimetic that selectively homes to vasculature ofpremalignant pancreas, the peptide or peptidomimetic containing at least5 contiguous amino acids of an amino acid sequence selected from CRSRKG(SEQ ID NO:9) and CEYQLDVE (SEQ ID NO:34), or a conservative variant orpeptidomimetic thereof, or (b) a peptide or peptidomimetic thatselectively homes to premalignant and malignant pancreatic vasculature,the peptide or peptidomimetic containing at least 5 contiguous aminoacids of an amino acid sequence selected from CRGRRST (SEQ ID NO:5) andFRVGVADV (SEQ ID NO:27), or a conservative variant or peptidomimeticthereof, thereby directing the moiety to the vasculature of thepancreatic premalignant lesion. In one embodiment, the moiety is atherapeutic moiety, such as an angiogenic inhibitor. In anotherembodiment, the moiety is a diagnostic moiety. Exemplary moieties usefulin this method are described herein below.

The invention also provides a method of directing a moiety to pancreatictumor cells and pancreatic tumor vasculature in an individual. Themethod involves administering to the individual a conjugate containing amoiety linked to: (a) a peptide or peptidomimetic that selectively homesto pancreatic tumor cells and pancreatic tumor vasculature, the peptideor peptidomimetic containing at least 5 contiguous amino acids of anamino acid sequence selected from CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQID NO:19), FRVGVADV and VGVGEWSV (SEQ ID NO:35), or a conservativevariant or peptidomimetic thereof, or (b) a peptide or peptidomimeticthat selectively homes to premalignant and malignant pancreaticvasculature, the peptide or peptidomimetic containing at least 5contiguous amino acids of an amino acid sequence selected from CRGRRST(SEQ ID NO:5) and FRVGVADV (SEQ ID NO:27), or a conservative variant orpeptidomimetic thereof, thereby directing the moiety to pancreatic tumorcells and pancreatic tumor vasculature.

Imaging

Selective delivery of diagnostic agents to vasculature that supportstumors provides a tool for diagnosis of early or late stage cancers,such pancreatic cancer. The pancreas is an organ of the digestive systemthat contains exocrine and endocrine components. The exocrine componentis a compound gland with branched ducts and serous secretory units. Theendocrine component synthesizes and secretes into the blood, hormonesthat regulate glucose, lipid and protein metabolism of the body. Thiscomponent is dispersed within the exocrine component as distinct cellmasses called islets of Langerhans. Islets of Langerhans are polygonalendocrine cells arranged in short, irregular cords that are profuselyinvested with a network of fenestrated capillaries. As is shown herein,a peptide or peptidomimetic of the invention can be used to distinguishbetween normal pancreatic vasculature and premalignant or malignantpancreatic vasculature. Therefore, when linked to a detectable moiety, apeptide or peptidomimetic of the invention can be used to visualize, orotherwise render detectable, early changes in pancreatic vascularassociated with a precancerous state, as well as later changes inpancreatic vascular associated with a more advanced or malignant state.

The invention provides a method for imaging pancreatic premalignantlesions in an individual. The method involves: (a) administering to theindividual a conjugate containing a detectable moiety linked to apeptide or peptidomimetic that selectively homes to vasculature ofpremalignant pancreas, the peptide or peptidomimetic containing at least5 contiguous amino acids of an amino acid sequence selected from CRSRKG(SEQ ID NO:9) and CEYQLDVE (SEQ ID NO:34), or a conservative variant orpeptidomimetic thereof, and (b) detecting the conjugate, thereby imagingpancreatic premalignant lesions.

The invention also provides a method of imaging pancreatic tumors andpancreatic tumor vasculature in an individual. The method involves (a)administering to the individual a conjugate containing a detectablemoiety linked to a peptide or peptidomimetic that selectively homes topancreatic tumor cells and pancreatic tumor vasculature, the peptide orpeptidomimetic containing at least 5 contiguous amino acids of an aminoacid sequence selected from CKAAKNK (SEQ ID NO:15) and CKGAKAR (SEQ IDNO:19), or a conservative variant or peptidomimetic thereof, and (b)detecting the conjugate, thereby imaging the pancreatic tumors andpancreatic tumor vasculature.

As used herein, the term “detectable moiety” means any molecule that canbe administered in vivo and subsequently detected. Exemplary detectablemoieties useful in the conjugates and methods of the invention include,without limitation, radiolabels and fluorescent molecules. Exemplaryradionuclides include indium-111, technetium-99, carbon-11, andcarbon-13. Fluorescent molecules include, without limitation,fluorescein, allophycocyanin, phycoerythrin, rhodamine, and Texas red.

The methods of the invention for imaging the vasculature of apremalignant tissue such as premalignant pancreas, or for imaging oftumor cells and tumor vasculature, such as pancreatic tumor cells andpancreatic rumor vasculature can be useful for early detection ofpremalignant lesions or malignancies including but not limited to,pancreatic premalignant lesions and tumors. Following administration ofa conjugate of the invention containing a detectable moiety, thevasculature of premalignant tissue or tumor tissue is visualized. If theimage is positive for the presence of such vasculature, furtherevaluation can be performed for the size of the tumor, if any, and thequantity of vascular infiltration. These results provide valuableinformation to the clinician with regard to the stage of development ofthe cancer and the presence or probability of metastasis. It isunderstood that the methods of the invention are application to avariety of types of premalignant lesions and cancers of organsincluding, yet not limited to, cancers of digestive tract, such as headand neck cancers, esophageal cancer, stomach cancer, pancreatic cancer,liver cancer,

colon and rectal cancer, anal cancer; cancers of genital and urinarysystems, such as kidney cancer, bladder cancer, testicular cancer,prostate cancer; cancers of the nervous system, such as brain cancer;bone cancer; nasopharyngeal cancer; retroperitoneal sarcomas; softtissue cancers; thyroid cancer; breast cancer; ovarian cancer;gynecological cancers; choriocarcinoma and other types of cancers.

In a method of the invention for imaging vasculature of a premalignanttissue, the conjugate administered contains a detectable moiety thatallows detection or visualization of the vasculature of the premalignanttissue such as a pancreatic premalignant lesion. In a method of theinvention for imaging calls and vasculature of a malignant tissue, theconjugate administered contains a detectable moiety that allowsdetection or visualization of pancreatic tumors and pancreatic tumorvasculature. For such in vivo diagnostic imaging, a peptide orpeptidomimetic is linked to a detectable moiety that, uponadministration to the subject, is detectable external to the subject.Such a detectable moiety can be, for example, a gamma ray emittingradionuclide such as indium-113, indium-115 or technetium-99; followingadministration to a subject, the conjugate can be visualized using asolid scintillation detector.

A detectable moiety useful in the invention can be, for example, aparamagnetic ion such as a magnesium, manganese, iron oxide, dysprosiumor gadolinium ion. Exemplary iron oxides include dextran-coatedsuperparamagnetic iron oxide, carboxydextran-coated superparamagneticiron oxide, ultrasmall superparamagnetic iron oxide, monocrystallineiron oxide nanocompound and ferromagnetic iron lignosulfonate. Exemplarygadolinium-based paramagnetic moieties include Gd-DTPA, dextran-Gd-DTPA,Gd-DTPA-24-cascade-polymer, Gd-DTPA-polylysine, Gd-melanin polymer and6-dendrimer-Gd-DTPA. When linked to a peptide or peptidomimetic of theinvention, such a paramagnetic ion can be used for enhanced magneticresonance imaging (MRI). A paramagnetic ion can be linked directly to apeptide or peptidomimetic or linked indirectly, for example, by beingcontained in a liposome that is linked to the peptide or peptidomimetic.

A detectable moiety also can be an agent that facilitates detection ofpremalignant and malignant tissue in vitro. For example, a conjugate cancontain a peptide or peptidomimetic of the invention linked to anenzyme, which produces a visible signal when an appropriate substrate ispresent. A detectable moiety useful in such a conjugate can be, forexample, alkaline phosphatase or luciferase or the like, and can bedetected by immunohistochemistry using routine techniques.

Therapy

The invention provides a method of treating a pancreatic premalignantlesion in an individual. The method involves administering to theindividual a conjugate containing a therapeutic moiety linked to: (a) apeptide or peptidomimetic that selectively homes to premalignant andmalignant pancreatic vasculature, the peptide or peptidomimeticcontaining at least 5 contiguous amino acids of an amino acid sequenceselected from CRGRRST (SEQ ID NO:5), and FRVGVADV (SEQ ID NO:27), or aconservative variant or peptidomimetic thereof, or (b) a peptide orpeptidomimetic that selectively homes to vasculature of premalignantpancreas containing at least 5 contiguous amino acids of an amino acidsequence selected from CRSRKG (SEQ ID NO:9) and CEYQLDVE (SEQ ID NO:34),thereby directing the therapeutic moiety to the pancreatic premalignantlesion in the individual to treat the pancreatic premalignant lesion. Inone embodiment, the moiety is a therapeutic moiety, such as anangiogenic inhibitor. In one embodiment, the peptide or peptidomimeticselectively homes to vasculature of premalignant pancreas.

The invention also provides a method of reducing the severity ofpancreatic cancer in an individual. The method involves administering tothe individual a conjugate containing a therapeutic moiety linked to:(a) a peptide or peptidomimetic that selectively homes to pancreatictumor cells and pancreatic tumor vasculature, the peptide orpeptidomimetic containing at least 5 contiguous amino acids of an aminoacid sequence selected from CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ IDNO:19), FRVGVADV and VGVGEWSV (SEQ ID NO:35), or a conservative variantor peptidomimetic thereof, or (b) a peptide or peptidomimetic thatselectively homes to premalignant and malignant pancreatic vasculature,the peptide or peptidomimetic containing at least 5 contiguous aminoacids of an amino acid sequence selected from CRGRRST (SEQ ID NO:5) andFRVGVADV (SEQ ID NO:27), or a conservative variant or peptidomimeticthereof, thereby directing the therapeutic moiety to pancreatic tumorcells or pancreatic tumor vasculature in the individual to reduce theseverity of the pancreatic cancer.

Therapeutic Moieties

A variety of therapeutic moieties are useful in the conjugates andmethods of the invention, including, without limitation, anti-angiogenicagents and cytotoxic agents, such as those that target a DNA-associatedprocess. As used herein, the term “therapeutic moiety” is used broadlyto mean a physical, chemical, or biological material that can be linkedto a homing molecule and that alters biological activity in a normal orpathologic tissue upon administration. A therapeutic moiety, therefore,is potentially useful for the treatment of disease conditions. Atherapeutic moiety can be any natural or nonnatural material including abiological material, such as a cell or phage; an organic chemical, suchas a small molecule; a radionuclide; a nucleic acid molecule oroligonucleotide; a polypeptide; or a peptide or peptidomimetic.Therapeutic moieties useful in the invention include, withoutlimitation, anti-angiogenic agents; cancer chemotherapeutic agents;cytotoxic agents; pro-apoptotic agents. A therapeutic moiety useful inthe invention can be expressed on, contained in, or linked to any of thefollowing: phage or other virus, cell, liposome, polymeric ornon-polymeric matrix, gold or other particle, or a microdevice,nanodevice, or nano-scale semiconductor material. These and othermaterials known in the art can be components of the conjugates of theinvention.

A therapeutic moiety useful in a conjugate of the invention can be, forexample, an anti-angiogenic agent. As used herein, the term“anti-angiogenic agent” means a molecule that reduces or inhibitsangiogenesis. An anti-angiogenic agent useful in the conjugates andmethods of the invention can be, for example, an inhibitor orneutralizing antibody that reduces the expression or signaling of anangiogenic factor such as vascular endothelial growth factor (VEGF),which is a major inducer of angiogenesis in normal and pathologicalconditions, and is essential in embryonic vasculogenesis. The biologicaleffects of VEGF include stimulation of endothelial cell proliferation,survival, migration and tube formation, and regulation of vascularpermeability. An anti-angiogenic agent also can inhibit anotherangiogenic factor such as a member of the fibroblast growth factor (FGF)family such as FGF-1 (acidic), FGF-2 (basic), FGF-4 or FGF-5 (Slavin etal., Cell Biol. Int. 19:431-444 (1995); Folkman and Shing, J. Biol.Chem. 267:10931-10934 (1992)) or angiopoietin-1, a factor that signalsthrough the endothelial cell-specific Tie2 receptor tyrosine kinase(Davis et al., Cell 87:1161-1169 (1996); and Suri et al., Cell87:1171-1180 (1996)), or the receptor of one of these angiogenicfactors. It is understood that a variety of mechanisms can act toinhibit activity of an angiogenic factor including, without limitation,direct inhibition of receptor binding or of secretion of the angiogenicfactor into the extracellular space, and inhibition of signaling,expression or function of the angiogenic factor.

A variety of anti-angiogenic agents useful in the invention are known inthe art and can be prepared by routine methods. See, for example,Hagedorn and Bikfalvi, Crit. Rev. Oncol. Hematol. 34:89-110 (2000) andKirsch et al., J. Neurooncol. 50:149-163 (2000). Anti-angiogenic agentsinclude, without limitation, small molecules; proteins such asangiogenic factors and receptors, transcription factors, and antibodiesand antigen-binding fragments thereof; peptides and peptidomimetics; andnucleic acid molecules including ribozymes, antisense oligonucleotides,and nucleic acid molecules encoding, for example, dominant negativeangiogenic factors and receptors, transcription factors, and antibodiesand antigen-binding fragments thereof. Exemplary anti-angiogenic agentsuseful in the conjugates and methods of the invention include, yet arenot limited to, angiostatin, endostatin, metastatin and 2ME2 (EntreMed;Rockville, Md.); anti-VEGF antibodies such as Avastin (Genentech; SouthSan Francisco, Calif.); VEGFR-2 inhibitors such as the small moleculesSU5416 and SU6668, (SUGEN; South San Francisco, Calif.); heparin-bindingfragments of fibronectin; modified forms of antithrombin; collagenaseinhibitors; basement membrane turnover inhibitors; angiostatic steroids;platelet factor 4, and fragments and peptides thereof; thrombospondin,and fragments and peptides thereof; and doxorubicin (O'Reilly et al.,Cell 79:315-328 (1994)); O'Reilly et al., Cell 88: 277-285 (1997);Homandberg et al., Am. J. Path. 120:327-332 (1985); Biochim. Biophys.Acta 874:61-71 (1986); and O'Reilly et al., Science 285:1926-1928(1999)). It is understood that these as well as other anti-angiogenicagents known in the art or that can be prepared by routine methods areencompassed by the term “anti-angiogenic agent” and can be used in thevarious conjugates and methods of the invention.

It is understood by those skilled in the art that an anti-angiogenicagent can be particularly efficacious when targeted to a specific stageof tumor progression (see, for example, Bergers et al., Science284:808-812 (1999); and Bergers et al., J. Clin. Invest. 111:1287-1295(2003)). Thus, in one embodiment, an anti-angiogenic agent useful in theinvention is “effective against premalignant vasculature.” As usedherein, the term “anti-angiogenic agent effective against premalignantvasculature” means an angiogenic agent that can significantly reduce thenumber of angiogenic lesions during the premalignant phase ofcarcinogenesis, before solid tumors have formed. Such an anti-angiogenicagent is an anti-angiogenic agent effective against premalignantvasculature whether or not the agent also significantly reduces tumorburden or extends life-span in animals with tumors, including animalswith small solid tumors or animals having large tumors and end-stagedisease. As non-limiting examples, an anti-angiogenic agent effectiveagainst pre-malignant vasculature can be BB-94 (batimastat), abroad-spectrum inhibitor of matrix metalloproteinases (Talbot and Brown,Eur. J. Cancer 32A: 2528 (1996)); SU5416 (SUGEN), a small moleculeinhibitor of VEGFR-2; or endostatin, a carboxy-terminal fragment ofcollagen XVIII (O'Reilly et al., Cell 88: 277 (1997); and Boehm et al.,Nature 390: 404 (1997)), alone or combined with angiostatin, an internalfragment of plasminogen (O'Reilly et al., Cell 79: 314 (1994); andO'Reilly et al., Nature Med. 2: 689 (1996)). See, also, Bergers et al.,supra, 1999; Bergers et al., supra, 2003.

An anti-angiogenic agent useful in the invention also can be ananti-angiogenic agent effective against tumor vasculature. As usedherein, the term “anti-angiogenic agent effective against tumorvasculature” means an angiogenic agent that can significantly reducetumor burden or extend life-span of animals having solid, vascularizedtumors. Such an anti-angiogenic agent is an anti-angiogenic agenteffective against tumor vasculature if there is efficacy against one ormore of the following: small solid tumors, tumors with well-definedmargins, invasive tumors or end-stage cancer, whether or not the agentsignificantly reduces the number of angiogenic lesions during thepre-malignant phase of carcinogenesis. As non-limiting examples, ananti-angiogenic agent effective against tumor vasculature can beefficacious only against small vascularized tumors, or against largetumors as well as small vascularized tumors. An anti-angiogenic agenteffective against tumor vasculature can be, without limitation, ananti-angiogenic agent such as BB-94 (batimastat), endostatin, orangiostatin, which is effective against small tumors without significantefficacy on the large tumors characteristic of end-stage cancer (Bergerset al., supra, 1999). An anti-angiogenic agent effective against tumorvasculature further can be an anti-angiogenic agent effective againstsmall tumors as well as large tumors in animals with short lifeexpectancy such as, without limitation, AGM-1470 (TNP470), a smallmolecule inhibitor of endothelial cell proliferation (Ingber et al.,Nature 348:555 (1990); Griffith et al., Chem. Biol. 4:461 (1997); Sin etal., Proc. Natl. Acad. Sci. U.S.A. 94:6099 (1997); and Castronovo andBelotti, Eur. J. Cancer 32A:2520 (1996)).

A therapeutic moiety useful in a conjugate of the invention can be, forexample, a cytotoxic agent. As used herein, the term “cytotoxic agent”means any molecule that results in cell death by any mechanism.Exemplary cytotoxic agents useful in a conjugate of the inventionencompass, without limitation, taxanes such as docetaxel; anthracyclinssuch as doxorubicin; alkylating agents; vinca alkaloids;anti-metabolites; platinum agents such as cisplatin or carboplatin;steroids such as methotrexate; antibiotics such as adriamycin;antimicrobial peptides, described herein below; and other cancerchemotherapeutic agents, which are chemical agents that inhibit theproliferation, growth, life-span or metastatic activity of cancer cells.

Effective cytotoxic agents useful in the invention include those thattarget DNA, for example, alkylating agents, agents that intercalate intoDNA, and agents that result in double-stranded DNA breaks. ExemplaryDNA-targeted drugs include, without limitation, cyclophosphamide,melphalan, mitomycin C, bizelesin, cisplatin, doxorubicin, etoposide,mitoxantrone, SN-38, Et-743, actinomycin D, bleomycin, TLK286 and SGN-15(Hurley, supra, 2002). It is understood that DNA-targeting cytotoxicagents can be particular useful when combined in conjugates with ahoming molecule that localizes, at least in part, to the nuclei ofcells. Thus, in one embodiment the invention provides a conjugatecontaining a therapeutic moiety linked to a peptide or peptidomimeticthat selectively homes to vasculature of premalignant pancreas andcontains at least 5 contiguous amino acids of an amino acid sequenceselected from CRSRKG (SEQ ID NO:9) and CEYQLDVE (SEQ ID NO:34), or aconservative variant or peptidomimetic thereof, where the therapeuticmoiety is a cytotoxic agent that targets a DNA-associated process. In afurther embodiment, the invention provides a conjugate containing atherapeutic moiety linked to a peptide or peptidomimetic thatselectively homes to pancreatic tumor cells and pancreatic tumorvasculature and contains at least 5 contiguous amino acids of an aminoacid sequence selected from CKAAKNK (SEQ ID NO:15), CKGAKAR (SEQ IDNO:19), and VGVGEWSV (SEQ ID NO:35), or a conservative variant orpeptidomimetic thereof. Useful cytotoxic agents that target aDNA-associated process include, without limitation, alkylating agents,anti-tumor antibiotics and sequence-selective agents and furtherencompass agents such as cyclophosphamide, melphalan, mitomycin C,bizelesin, cisplatin, doxorubicin, etoposide, mitoxantrone, SN-38,Et-743, actinomycin D, bleomycin and TLK286.

Taxanes are cytotoxic agents useful in a conjugate of the invention.Useful taxanes include, without limitation, docetaxel (Taxotere; AventisPharmaceuticals, Inc.; Parsippany, N.J.) and paclitaxel (Taxol;Bristol-Myers Squibb; Princeton, N.J.). See, for example, Chan et al.,J. Clin. Oncol. 17:2341-2354 (1999), and Paridaens et al., J. Clin.Oncol. 18:724 (2000).

A cytotoxic agent useful in a conjugate of the invention also can be ananthracyclin such as doxorubicin, idarubicin or daunorubicin.Doxorubicin is a commonly used cancer chemotherapeutic agent (Stewartand Ratain, In: “Cancer: Principles and practice of oncology” 5th ed.,chap. 19 (eds. DeVita, Jr., et al.; J. P. Lippincott 1997); Harris etal., In “Cancer: Principles and practice of oncology,” supra, 1997). Inaddition, doxorubicin has anti-angiogenic activity that can contributeto its effectiveness in treating cancer (Folkman, supra, 1997; Steiner,In “Angiogenesis: Key principles-Science, technology and medicine,” pp.449-454 (eds. Steiner et al.; Birkhauser Verlag, 1992)).

An alkylating agent such as melphalan or chlorambucil also can be acytotoxic agent useful in a conjugate of the invention. Similarly, avinca alkaloid such as vindesine, vinblastine or vinorelbine; or anantimetabolite such as 5-fluorouracil, 5-fluorouridine or a derivativethereof can be a cytotoxic agent that can be linked to a homing moleculein a conjugate of the invention.

Cytotoxic agents useful in the conjugates of the invention also include,without limitation, platinum agents. Such a platinum agent can be, forexample, cisplatin or carboplatin as described, for example, in Crown,Seminars in Oncol. 28:28-37 (2001). Other cytotoxic agents useful in aconjugate of the invention include, but are not limited to,methotrexate, mitomycin-C, adriamycin, ifosfamide and ansamycins.

A cytotoxic agent also can be, for example, an antimicrobial peptide. Inone embodiment, the invention provides a conjugate in which a peptide orpeptidomimetic that selectively homes to vasculature of premalignantpancreas that is linked to an antimicrobial peptide, where the conjugateis selectively internalized by vasculature of a As used herein, the term“antimicrobial peptide” means a naturally occurring or synthetic peptidehaving antimicrobial activity, which is the ability to kill or slow thegrowth of one or more microbes and that has low mammalian cell toxicitywhen not linked to a homing molecule. An antimicrobial peptide can killor slow the growth of, for example, one or more strains of bacteriaincluding a Gram-positive or Gram-negative bacteria, or a fungi orprotozoa. Thus, an antimicrobial peptide can have, for example,bacteriostatic or bacteriocidal activity against, for example, one ormore strains of Escherichia coli, Pseudomonas aeruginosa orStaphylococcus aureus. While not wishing to be bound by the following,an antimicrobial peptide can have biological activity due to the abilityto form ion channels through membrane bilayers as a consequence ofself-aggregation.

An antimicrobial peptide is typically highly basic and can have a linearor cyclic structure. As discussed further below, an antimicrobialpeptide can have an amphipathic α-helical structure (see U.S. Pat. No.5,789,542; Javadpour et al., J. Med. Chem. 39:3107-3113 (1996); andBlondelle and Houghten, Biochem. 31: 12688-12694 (1992). Anantimicrobial peptide also can be, for example, a β-strand/sheet-formingpeptide as described in Mancheno et al., J. Peptide Res. 51:142-148(1998).

An antimicrobial peptide can be a naturally occurring or syntheticpeptide. Naturally occurring antimicrobial peptides have been isolatedfrom biological sources such as bacteria, insects, amphibians, andmammals and are thought to represent inducible defense proteins that canprotect the host organism from bacterial infection. Naturally occurringantimicrobial peptides include the gramicidins, magainins, mellitins,defensins and cecropins (see, for example, Maloy and Kari, Biopolymers37:105-122 (1995); Alvarez-Bravo et al., Biochem. J. 302:535-538 (1994);Bessalle et al., FEBS 274:151-155 (1990); and Blondelle and Houghten inBristol (Ed.), Annual Reports in Medicinal Chemistry pages 159-168Academic Press, San Diego)). As discussed further below, anantimicrobial peptide also can be an analog of a natural peptide,especially one that retains or enhances amphipathicity.

An antimicrobial peptide incorporated within a conjugate of theinvention has low mammalian cell toxicity when not linked to a homingmolecule of the invention. Mammalian cell toxicity readily can beassessed using routine assays. For example, mammalian cell toxicity canbe assayed by lysis of human erythrocytes in vitro as described inJavadpour et al., supra, 1996. An antimicrobial peptide having lowmammalian cell toxicity is not lytic to human erythrocytes or requiresconcentrations of greater than 100 μM for lytic activity, preferablyconcentrations greater than 200, 300, 500 or 1000 μM for lytic activity.

It is understood by one skilled in the art of medicinal oncology thatthese and other agents are useful therapeutic moieties, which can beused separately or together in the conjugates and methods of theinvention. It further is understood that a conjugate of the inventioncan contain one or more of such therapeutic moieties and that additionalcomponents can be included as part of the conjugate, if desired. As anexample, in some cases it can be desirable to utilize an oligopeptidespacer between the homing molecule and the therapeutic agent(Fitzpatrick and Garnett, Anticancer Drug Des. 10:1-9 (1995)).

Staging Tumor Progression

The invention provides a method of staging tumor progression in anindividual having or suspected of having a pancreatic premalignantlesion or pancreatic tumor. The method involves: (a) administering tothe individual at least one conjugate containing a detectable moietylinked to (i) a peptide or peptidomimetic that selectively homes tovasculature of premalignant pancreas, the peptide or peptidomimeticspecifically binding a cognate receptor for CRSRKG (SEQ ID NO:9) orCEYQLDVE (SEQ ID NO:34), or (ii) a peptide or peptidomimetic thatselectively homes to pancreatic tumor cells and pancreatic tumorvasculature, the peptide or peptidomimetic specifically binding acognate receptor for an amino acid sequence selected from CKAAKNK (SEQID NO:15), CKGAKAR (SEQ ID NO:19) and VGVGEWSV (SEQ ID NO:35); and (b)detecting the conjugate, wherein detection of the conjugate containing apeptide or peptidomimetic that selectively homes to vasculature ofpremalignant pancreas indicates a premalignant stage of tumorprogression in the individual and wherein detection of the conjugatecontaining a peptide or peptidomimetic that selectively homes topancreatic tumor cells and pancreatic tumor vasculature indicates amalignant stage of tumor progression in the individual. Exemplarymoieties useful in this method are described herein above.

A stage of a tumor refers to the degree of progression of a tumor. Apremalignant stage of tumor progression in a tissue means precancerousstate of the tissue, which can be characterized, for example, byabnormal tissue structural and functional changes in comparison to anormal state of the tissue. Such changes are described in the art, forexample, as metaplasia, dysplasia, hyperplasia, carcinoma in situ,angiogenic and the like, depending on the degree of structural and/orfunctional changes. Histological and biochemical changes characteristicof precancerous tissues depend upon the type of tumor and can bedetermined by those skilled in the art. A malignant stage of tumorprogression means that the tumor is established, as indicated by, forexample, increased proliferation rate compared to normal. Various stagesof tumor development are well known to those of skill in the art, asexemplified in Markman, “Basic Cancer Medicine,” Saunders, (ed. Zorab,R.) (1997). For example, malignant cancers can be staged into threegeneral stages—localized, regional spread, and distant spread. Cancersalso can be staged using the TNM system, which considers the extent ofdirect spread within affected and nearby tissues, the extent of spreadto nearby lymph nodes, and the extent of spread to distant organs. Basedon these features, spread of cancers can be summarized by assigningRoman numerals from 0 through IV. Those skilled in the art can select anappropriate staging system for a particular type of cancer.

Routes of Administration

It is understood that a variety of routes of administration are usefulin the methods of the invention. Such routes encompass systemic andlocal administration and include, without limitation, oraladministration, topical application, intravenous injection,intraperitoneal injection, intramuscular injection, subcutaneousinjection, transdermal diffusion or electrophoresis, local injection,and extended release delivery devices including locally implantedextended release devices such as bioerodible or reservoir-basedimplants.

The following examples are intended to illustrate but not limit thepresent invention.

Example I Isolation of Stage-Specific Phage

This example describes the isolation of phage that selectively home topremalignant lesions and malignant tumors.

To isolate peptides that selectively home to premalignant lesions andmalignant tumors, the RIP1-Tag2 mouse model was used. RIP1-Tag2 micedevelop multifocal angiogenic islet progenitors and then solid tumors ina stepwise manner, such that at 12 weeks of age, each mouse typicallyhas approximately 50 angiogenic islets and 2-6 small tumors. Using 12week-old mice, phage that bind to angiogenic islet progenitors and/ortumors in the same mouse were identified.

The generation of RIP1-Tag2 mice is described, for example, in Hanahan,Nature 315:115-122 (1985). Angiogenic islets were isolated from 8 and 12week old RIP1-Tag2 mice by collagenase digestion of the excisedpancreas, and selected based on their red, hemorrhagic appearance(Parangi et al. Cancer Res. 55: 6071-6076 (1995)). Tumors weremicrodissected from the excised pancreas of 12-week old RIP1-Tag2 miceand the surrounding exocrine tissue carefully removed. The synchronicityof tumorigenesis in the RIP1-Tag2 model allowed for simultaneousisolation of angiogenic islets and tumors from the same mouse at 12weeks of age, such that homing of individual phage to different stagesin tumor progression in an individual mouse/pancreas could be compared.

In order to enrich for phage that bind to RIP1-Tag2 target cells(endothelial, perivascular and tumor cells), a pre-selection step wasperformed on cell suspensions prepared from premalignant and malignantpancreatic lesions. Pre-selection methods are described, for example, inLaakkonen et al. Nat. Med. 8:751-755 (2002) and Porkka et al. Proc.Natl. Acad. Sci. USA 99, 7444-7449 (2002). The pre-selection stepinvolved two rounds of ex vivo selection from a CX₇C peptide library oncell suspensions from angiogenic islets or solid tumors. For the ex vivoselections, cell suspensions were prepared from the different RIP1-Tag2lesions in 12 week old RIP1-Tag2 mice and incubated overnight at 4° C.with 10⁹ plaque forming units (p.f.u.) of a T7 phage (Novagen)displaying the CX₇C peptide library. The cells were washed to removeunbound phage, and bound phage were rescued and amplified in E. coli.This procedure resulted in enrichment in phage that bound to tumor,endothelial and other stromal cells present in the suspension.

Ex vivo screening yielded phage pools that bound 7- to 8-fold over acontrol, non-recombinant phage to their respective target cells (seeFIG. 1A). These enriched phage pools were used in subsequent in vivorounds to select for phage that homed specifically to either angiogenicislets or tumors in RIP1-Tag2 mice.

For in vivo selection of phage that home specifically to premalignantlesions and malignant tumors, the ex vivo pre-selected phage pool wasinjected intravenously into 12 week old RIP1-Tag2 mice through the tailvein, allowed to circulate for 7 minutes and heart-perfused with PBS toremove unbound intravascular phage. As the vasculature is preferentiallyavailable for the phage to bind in this selection, there is anenrichment of phage that bind to the endothelium of the target tissue.

The RIP1-Tag2 lesions and control tissues (brain, kidney, spleen, lung,‘white’ pancreas lacking hemorrhagic lesions and liver were excised toallow for comparison of homing efficiencies. Cell suspensions wereprepared by mechanical disruption of the tissues. Tissues were washed toremove unbound phage, and the bound phage rescued and amplified byadding E. coli. The phage pool was re-injected into mice at a similardisease stage, and the cycle repeated. In each experiment,non-recombinant control phage were used as a control for relativeselectivity.

Three rounds of in vivo selection on angiogenic islets resulted in aphage pool that selectively homed to angiogenic islets. The homing toangiogenic islets was 7-fold higher than to tumors in the same mouse(FIG. 1B). There was no homing to control organs. The tumor selectionyielded a pool that showed an 8-fold preference for tumors versusangiogenic islets in the same mouse following two rounds of in vivoselection (FIG. 1C).

Sets of 96 phage clones were randomly collected from each homing phagepopulation that selectively homed to premalignant angiogenic islets orpancreatic tumor cells. Peptide-encoding DNA inserts from collectedphage clones were amplified by PCR, and the PCR products sequenced.Phage representing the most frequently appearing peptide motifs wereindividually tested for their ability to selectively home to the lesionson which they were selected, relative to other stages in thetumorigenesis pathway and to control organs. Six of the phage selectedfor further analysis were from the tumor screen (referred to as KAA,RGR, RSR, VGVA, VGVG and KAR), and one (EYQ) was picked from theangiogenic screen. Peptide sequences corresponding to each of thesepeptide motifs are shown in Table 1.

The identified RIP1-Tag2 homing phage fell into three classes based ontheir ability to home either to angiogenic islets or to tumors in vivo(FIG. 1D) and their ex vivo binding patterns. The identified classeswere tumor-selective phage (KAA, KAR and VGVG); angiogenicislet-selective phage (RSR and EYQ); and phage that home to both typesof lesion (VGVA and RGR) (See Table 1 for peptide sequences). Some ofthe selected peptides that share similar peptide motifs also displaysimilar homing patterns. For example, KAA and KAR (CKAAKNK andCKGAKAR=XBXXBXB, where B represents basic residues and X denotesuncharged residues) both preferentially home to tumors over angiogenicislets. However, other related peptides such as RGR and RSR(CRGRRST andCRSRKG=XBXBBX) have quite different homing capabilities.

TABLE I Peptide sequence Extended motif Mouse protein Accession Peptide(SEQ ID NO: ) (SEQ ID NO: ) with the motif number RGR CRGRRST (5) RGRRS(2) PDGF-B P31240 RGRR (1) Stromal interaction Q9P246 molecule 2 RSRCRSRKG (9) CRSR-G (38) Cadherin EGF 035161 LAG receptor 1 KAA CKAAKNK(15) CKA-K (39) WNT-2 NP076142 KAR CKGAKAR (19) CKGAKA (20) Collagen XIIQ60847 AKAR (16) Collagen XII Q60847 GAKAR (17) Claudin 9 Q9Z0S7 VGVAFRVGVADV (27)) F-VGVADV (40) Collagen XII Q60847 RVGV (21) Collagen XIIQ60847 EYQ CEYQLDVE (34) CEYQL (28) Semaphorin 4C Q64151 YQLDV (32)FGFR-1 P16092 YQLDV (32) Tie-1 Q06806 Table 1. Candidate mouse proteinssharing motifs with peptides. Peptides were analyzed using a BLAST(NCBI) search against the SWISSPROT database, using the option for shortnearly exact matches, to identify mouse proteins with homologoussequences.

Thus, a combination of ex vivo and in vivo phage screening was used toobtain peptides that selectively home to either premalignant pancreatictissue, malignant pancreatic tissue or both premalignant and malignantpancreatic tissue.

Example II Tumor Stage-Specific Homing of Fluorescein-ConjugatedPeptides In Vivo

This example describes that selective phage homing was due to thedisplayed peptide.

To confirm that selective phage homing was due to the displayed peptidesequences, localization of fluorescein-conjugated peptides afterintravenous injection was observed. For this analysis, one peptide fromeach homing class was selected as follows: CRSRKG (SEQ ID NO:9),referred to as RSR (angiogenic-selective), CKAAKNK (SEQ ID NO:15),referred to as KAA (tumor-selective) and CRGRRST (SEQ ID NO:5), referredto as RGR (angiogenic- and tumor-homing). Eight week old RIP1-Tag2 micewere used to examine peptide localization during the angiogenic switch,and 12-week old RIP1-Tag2 mice were used to visualize both angiogenicislets and tumors.

Fluorescein-conjugated peptides corresponding to phage insert sequenceswere synthesized using an automated peptide synthesizer with standardsolid-phase fluorenylmethoxycarbonyl (Fmoc) chemistry. 100 mg of eachindividual fluorescein-conjugated peptide was injected intravenouslyinto the tail vein of RIP1-Tag2 mice at 8 or 12 weeks of age, and intonormal BL/6 mice. The peptide was allowed to circulate for 7 minutes,followed by heart perfusion first with PBS and then with Zn-bufferedformalin. The RIP1-Tag2 pancreas and control organs (brain, kidney,liver, lung and spleen) were removed, fixed for one hour in formalin,washed with 1×PBS, placed in 30% sucrose for several hours, washed with1×PBS, and embedded in OCT (Tissue-Tek). Each peptide was injected intoat least three individual RIP1-Tag2 or normal mice at each of thedifferent stages. To examine the localization of injectedfluorescein-conjugated peptides, frozen sections (10 mm thick) were cuton a cryostat, mounted in Vectashield Mounting Medium with DAPI (VectorLaboratories) and visualized under an inverted fluorescent microscope ora confocal microscope (Zeiss LSM 510 META).

FIG. 2 shows specific homing of RSR, KAA and RGR peptides to normalislets, angiogenic islets and tumors. Visualization of angiogenicislet-selective peptide RSR homing is shown in normal islet (A),angiogenic islet (B), and tumor (C). Visualization of tumor-selectivepeptide KAA homing is shown in normal islet (D), angiogenic islet (E),and tumor (F). Visualization of angiogenic islet- and tumor-selectivepeptide RGR homing is shown in (G) normal islet, (H) angiogenic islet,and (I) tumor. Control tissues from a RIP1-Tag2 mouse injected withfluorescein-conjugated RGR-peptide are shown in (J) kidney; (K) brain,and (L) liver. Similar absence of fluorescence in control tissues wasobserved for the other injected peptides, indicative of a lack of homingto control tissues.

As can be seen in FIG. 2, RSR shows abundant accumulation in RIP1-Tag2angiogenic islets, but little or no localization in tumors or normalislets. KAA shows abundant localization in RIP1-Tag2 tumors but littleor no localization in angiogenic islets, or normal islets. Finally, RGRlocalizes in both RIP1-Tag2 angiogenic islets and tumors but little orno localization in normal islets. Fluorescence detected in kidney wasassessed as non-specific, likely resulting from uptake from glomerularfiltrate (FIG. 2J). Unexpectedly RSR, which was selected from the tumorphage screening, preferentially bound to angiogenic islets. This resultindicates that the epitope bound by RSR is present both in tumors andangiogenic islets, but is more abundant in angiogenic islets. Theobserved peptide localization profiles in each case were similar tolocalization profiles of the cognate phage (compare FIG. 1D and FIG. 2),with each peptide falling into the same of the three homing classes. Inaddition, control peptides did not show specific homing to any of theRIP1-Tag2 lesional stages or to a number of normal tissues

Thus, selective homing was confirmed to be due to the displayed peptidesequences, rather than the phage.

Example III Co-localization of Fluorescein-Conjugated Peptides withVascular Markers in RIP1-Tag2 Premalignant and Malignant Lesions

This example describes co-localization of fluorescein-conjugatedpeptides with vascular markers.

To confirm that intravenous administration of phage libraries selectsfor phage carrying peptides that bind to endothelial molecules specificfor the target vasculature, tissues were collected following i.v.infusion with the various fluorescein-conjugated peptides, sectioned,and evaluated with endothelial cell markers.

For immunohistochemistry, frozen slides were pre-incubated with blockingbuffer (1×PNB from NEN Biosciences) for one hour, washed several timesin 1×PBS and incubated with the primary antibody of interest overnightat 4° C. The cell-specific antibodies used were rat monoclonalanti-mouse CD31 (1:200; BD Pharmingen), rat monoclonal anti-mouseMECA-32 (1:200; BD Pharmingen), rabbit polyclonal anti-mouse NG2 (1:200;Chemicon), and rat monoclonal anti-mouse PDGFRb (CD140b) (1:200;eBioscience). The corresponding secondary antibodies; Cy-3 donkeyanti-rabbit IgG and Rhodamine Red donkey anti-rat IgG (JacksonImmunoResearch), were used at a 1:200 dilution and incubated for onehour at room temperature. The following species-matched immunoglobulinswere used as negative controls; rabbit IgG (Vector Laboratories) and ratIgG (Jackson ImmunoResearch) at a 1:200 dilution. The slides were washedseveral times in 1×PBS and mounted in Vectashield Mounting Medium withDAPI (Vector Laboratories). Hematoxylin and eosin (H&E) staining wasperformed for histological grading of adjacent sections by standardmethods, and lesions were graded as previously described (Lopez andHanahan, 2002).

The primary analysis involved immunostaining with a mousepan-endothelial cell antigen (MECA-32) antibody that recognizes a dimerof 50-55 kDa protein subunits present on all endothelial cells (Hallmanet al., 1995; Leppink et al., 1989) (FIG. 3 B,C,H,I,N,O). Additionalanalyses involved immunostaining to reveal CD31/PE-CAM, or systemicinfusion of a fluorescent-labeled lectin that binds to the endotheliallumen. In addition, tissue sections from peptide-infused mice werestained with an antibody recognizing NG2, a marker of the neovascularpericytes (Schlingemann et al., 1990, 1991) (FIG. 3 E,F,K,L,Q,R).

RSR peptide localization in an angiogenic islet is shown in panel A andD (green), while co-staining for MECA-32 (red) and the merge are shownin panels B and C. Co-staining for NG2 (red) is shown in panel E, withthe merge in panel F. KAA peptide localization in a tumor is shown inpanels G and J (green), while co-staining for MECA-32 (red) and themerge are shown in panels H and I. Co-staining for NG2 (red) is shown inpanel K, with the merge in panel L. RGR peptide localization in anangiogenic islet is shown in panel M and P (green), while co-stainingfor MECA-32 (red) and the merge are shown in panels N and O. Co-stainingfor NG2 (red) is shown in panel Q, with the merge in panel R.

As is shown in FIG. 3, all three peptides (RSR, KAA, and RGR) show someco-localization both with endothelial cell and pericyte markers,indicating that each homes to and binds moieties associated with bothcell types (FIG. 3). There was no co-localization of these peptides withMECA-32 or NG2 in the adjacent exocrine pancreas or in normal pancreaticislets.

Homing of peptides representing all three classes of binding specificityto both pericytes and endothelial cells was unexpected. This resultsupports studies indicating that RIP1-Tag2 tumor vasculature is leaky,as evidenced by extensive micro-hemorrhaging (Parangi et al., 1995) andmorphometric analysis (Hashizume et al., 2000; Morikawa et al., 2002;Thurston et al., 1998), by indicating that a circulating phage pool canhave access to the extra-luminal vascular microenvironment, wherereceptors on pericytes and in the extracellular matrix can beaccessible. The ex vivo pre-selection step used to enrich for RIP1-Tag2specific targets similarly can select for non-luminal endothelialbinding partners.

Thus, this example shows that peptides that selectively home topremalignant, malignant or both premalignant and malignant pancreatictissue, co-localize with endothelial cell and pericyte markers.

Example IV Tumor Specificity of Homing Phage and Peptides

This example describes the specificity of in vivo phage homing toangiogenic islets and/or tumors in the pancreas, and to the angiogenicvasculature in other tumor types.

To determine whether phage identified to home to pancreatic tumors alsohome to other types of tumors, these phage were tested using animalshaving various types of tumors, including bTC3 subcutaneous tumor andMDA MB-435 subcutaneous tumor. bTC3 transplant tumors arise followingsubcutaneous inoculation of nude mice with cultured islet tumor-derived(bTC3) cells (Efrat et al., 1988). Since the vasculature of asubcutaneously grown bTC3 tumor derives from skin, we also testedanother subcutaneous transplant tumor, arising from inoculation of theMDA-MB-435 human breast carcinoma cell line. Finally, K14-HPV16 mice,another well-studied transgenic mouse model of cancer that developtumors of the squamous epithelial cells of the skin (Arbeit et al.,1994; Coussens et al., 1996), was used to compare RIP1-Tag2 islet tumorsto a tumor with similar multistage pathogenesis arising in a differenttissue.

Tumors were dissected from the ear or chest of K14-HPV16 mice. For thebTC3 allograft models, 10⁶ bTC3 tumor cells (Efrat et al., 1988) wereinoculated under the skin of the rear flank of nu/nu mice in a BALB/cbackground and allowed to grow until approximately 5 mm in size, andthen used for experimental analysis. MDA-MB-435 xenograft models weregenerated by inoculating 10⁶ tumor cells subcutaneously in the chest ofnu/nu Balb/c mice. Tumors were used for the homing/binding experimentsat 8-12 weeks after injection of the tumor cells.

FIG. 4A is bar graph showing homing efficiency of individual phage to apancreatic tumor in a RIP1-Tag2 mouse, a bTC3-derived subcutaneoustransplant tumor in a nude mouse, and a squamous cell carcinoma in aK14-HPV16 mouse.

FIG. 4B is a table summarizing the relative homing offluorescein-conjugated peptides to different tumor models. +++ indicatesstrong homing, as revealed by the fluorescent intensity of i.v. injectedpeptide, ++ indicates moderate homing, + indicates weak homing, −indicates absence of homing.

FIGS. 4C, D and E show representative images of fluorescein-conjugatedKAR peptide homing to a RIP1-Tag2 pancreatic islet tumor (C); a bTC3subcutaneous tumor (D); and an MDA subcutaneous tumor (E).

As indicated by FIG. 4A, the relative homing efficiencies in the varioustumor models of the phage from the RIP1-Tag2 tumor screen fall broadlyinto two categories: those that selectively home to RIP1-Tag2 tumors(KAA, RGR, VGVA), and those that show a more general homing to othertumors in addition to RIP1-Tag2 (VGVG, KAR). The phage homing data weresupported by i.v. injection of fluorescein-conjugated peptidescorresponding to the phage, as shown in FIGS. 4C, D and E.

Thus, this example shows that certain peptides selectively home topancreatic tumors while others selectively home to other types of tumorsin addition to pancreatic tumors.

Example V Identification of Receptors for RGR Peptides

This example describes the identification of candidate vascularreceptors for peptides by sequence homology comparisons.

The set of peptides that selectively home to angiogenic premalignantlesions in the pancreas were used in database searches to identify mouseproteins containing sequences homologous to peptide sequences. Table 1lists candidate proteins of interest that contain such homologies. Manyof the candidate proteins have been previously associated with thevasculature, and could correspond to putative ligands mimicked by thephage-displayed peptides. One protein, collagen XII, was found to sharehomology with two peptides; KAR (CKGAKAR) and VGVA (FRVGVADV). It isinteresting to note that collagen XII was also identified by geneexpression profiling as a gene that is over-expressed in tumorendothelial cells (St. Croix et al., 2000; andhttp://mendel.imp.univie.ac.at/SEQUENCES/TEMS/mainpgs/temtable.html).

Another homology was observed for the RGR peptide (CRGRRST).Specifically, as is shown in Table 1, the sequence RGRRS is contained inthe B chain of the pro-form of platelet-derived growth factor (PDGF-B),a known ligand for the transmembrane receptor tyrosine kinase PDGFRβ.The RGR sequence homology spans the pro-peptide cleavage site ofpro-PDGF-B (Johnsson et al., 1984). Therefore, PDGFRβ was considered acandidate receptor for the RGR peptide.

To confirm that the RGR peptide binds specifically to PDGFRβ, phagedisplaying this peptide were incubated with 293T cells overexpressingPDGFRβ. Recombinant 293T cells were prepared by transfecting withplasmids encoding PDGFRβ or VEGFR-2 (Borges et al., 2000) using Fugenetransfection reagent (Roche Diagnostics). Briefly, 10 μg of plasmid wasmixed with 700 μl of DMEM without serum and 30 μl of Fugene reagent, andincubated for 15 minutes at room temperature before adding the mixtureto the cells. Forty-eight hours post-transfection the cells weredetached from the culture plates using EDTA and washed once with PBS.Recombinant phage displaying the RSR, RGR peptides and controlnonrecombinant phage (about 1×10⁹ pfu) were incubated with thetransfected cells for 2 hours at 4° C., followed by 5 washes with 1% BSAin PBS to remove the unbound phage. The bound phage were rescued byadding bacteria, and the binding efficiencies were determined by plaqueassay.

The results of these studies are shown in FIG. 5. FIG. 5A is a bar graphshowing binding of RGR or RSR phage to 293 cells transfected with eitherPDGFRβ, VEGFR2, or non-transfected cells. FIG. 5B shows co-localizationof fluorescein-conjugated RGR-peptide (panel a, green) with the PDGFRβantibody (panel b, red) and merged images (panels c and d) in RIP1-Tag2.

As shown in FIG. 5, binding of RGR phage was 20-fold more efficient toPDGFRβ-transfected cells than non-transfected cells. In contrast, nobinding above background was detected toward cells transfected withvascular endothelial growth factor receptor 2 (VEGFR2) (FIG. 5A).Moreover, RSR phage, which has a peptide sequence similar to RGR,displayed no specific binding was observed either to PDGFRβ or VEGFR2transfected cells (FIG. 5A). The association of RGR with PDGFRβ wasconfirmed when intravenously injected fluorescein-conjugated RGR peptidewas shown to co-localize with PDGFRβ visualized by subsequent immunostaining of tissue sections from RIP1-Tag2 tumors. Merging of theRGR-FITC image (FIG. 5B, panel a) with the antibody staining for PDGFRβ(FIG. 5B, panel b) revealed almost complete co-localization (FIG. 5B,panels c and d). These results indicate that PDGFRβ is a receptor forthe RGR peptide.

Thus, this example shows that PDGFRβ is a receptor for the RGR peptide(CRGRRST).

All journal articles, references and patent citations provided above, inparentheses or otherwise, whether previously stated or not, areincorporated herein by reference in their entirety.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1. A peptide or peptidomimetic, having a length of less than 60 amino acid residues and comprising at least 5 contiguous amino acids of an amino acid sequence selected from CKGAKAR (SEQ ID NO:19), FRVGVADV (SEQ ID NO:27), CEYQLDVE (SEQ ID NO:34) and VGVGEWSV (SEQ ID NO:35), or a conservative variant or peptidomimetic thereof.
 2. The peptide or peptidomimetic of claim 1, wherein said peptide or peptidomimetic is a peptide.
 3. The peptide or peptidomimetic of claim 1, having a length of less than 40 amino acid residues.
 4. The peptide or peptidomimetic of claim 1, having a length of less than 20 amino acid residues.
 5. The peptide or peptidomimetic of claim 1, having a length of less than 10 amino acid residues.
 6. A conjugate, comprising a therapeutic moiety linked to a peptide or peptidomimetic that selectively homes to vasculature of premalignant pancreas, said peptide or peptidomimetic comprising at least 5 contiguous amino acids of the amino acid sequence CEYQLDVE (SEQ ID NO:34), or a conservative variant or peptidomimetic thereof, or wherein said peptide or peptidomimetic binds specifically to a cognate receptor for SEQ ID NO:34.
 7. The conjugate of claim 6, wherein said peptide or peptidomimetic has a length of less than 100 residues.
 8. The conjugate of claim 6, wherein said peptide or peptidomimetic has a length of less than 50 residues.
 9. The conjugate of claim 6, wherein said peptide or peptidomimetic has a length of less than 25 residues.
 10. The conjugate of claim 6, wherein said peptide or peptidomimetic is a peptide. 11-22. (canceled)
 23. A conjugate, comprising a therapeutic moiety linked to a peptide or peptidomimetic that selectively homes to pancreatic tumor cells and pancreatic tumor vasculature, said peptide or peptidomimetic comprising at least 5 contiguous amino acids of an amino acid sequence selected from CKGAKAR (SEQ ID NO:19) and VGVGEWSV (SEQ ID NO:35), or a conservative variant or peptidomimetic thereof, or wherein said peptide or peptidomimetic binds specifically to a cognate receptor for SEQ ID NO:19 or SEQ ID NO:35.
 24. The conjugate of claim 23, wherein said peptide or peptidomimetic has a length of less than 100 residues.
 25. The conjugate of claim 23, wherein said peptide or peptidomimetic has a length of less than 50 residues.
 26. The conjugate of claim 23, wherein said peptide or peptidomimetic has a length of less than 25 residues.
 27. The conjugate of claim 23, wherein said peptide or peptidomimetic is a peptide.
 28. (canceled)
 29. The conjugate of claim 23, wherein said peptide or peptidomimetic comprises at least 5 contiguous amino acids of CKGAKAR (SEQ ID NO:19) or a conservative variant or peptidomimetic thereof.
 30. The conjugate of claim 23, wherein said peptide or peptidomimetic comprises at least 5 contiguous amino acids of VGVGEWSV (SEQ ID NO:35) or a conservative variant or peptidomimetic thereof. 31-43. (canceled)
 44. The conjugate of claim 23, wherein said therapeutic moiety is an angiogenic inhibitor.
 45. The conjugate of claim 44, wherein said angiogenic inhibitor is selective for mature tumor vasculature.
 46. The conjugate of claim 23, wherein said therapeutic moiety is a cytotoxic agent. 47-54. (canceled)
 55. A conjugate, comprising a therapeutic moiety linked to a peptide or peptidomimetic that selectively homes to premalignant and malignant pancreatic vasculature, said peptide or peptidomimetic comprising at least 5 contiguous amino acids of an amino acid sequence selected from FRVGVADV (SEQ ID NO:27), or a conservative variant or peptidomimetic thereof, or wherein said peptide or peptidomimetic binds specifically to a cognate receptor for SEQ ID NO:27. 56-67. (canceled)
 68. The conjugate of claim 55, wherein said peptide or peptidomimetic has a length of less than 100 residues.
 69. The conjugate of claim 55, wherein said peptide or peptidomimetic has a length of less than 50 residues.
 70. The conjugate of claim 55, wherein said peptide or peptidomimetic has a length of less than 25 residues.
 71. The conjugate of claim 55, wherein said peptide or peptidomimetic is a peptide.
 72. The conjugate of claim 55, wherein said therapeutic moiety is an angiogenic inhibitor.
 73. The conjugate of claim 72, wherein said angiogenic inhibitor is selective for mature tumor vasculature.
 74. The conjugate of claim 55, wherein said therapeutic moiety is a cytotoxic agent.
 75. A multivalent conjugate, comprising a therapeutic moiety linked to at least two peptides or peptidomimetics that selectively home to vasculature of premalignant pancreas, each of said peptides or peptidomimetics comprising at least 5 contiguous amino acids of the amino acid sequence CEYQLDVE (SEQ ID NO:34), or wherein each of said peptides or peptidomimetics binds specifically to a cognate receptor for SEQ ID NO:34; wherein said at least two peptides or peptidomimetics selectively home to pancreatic tumor cells and pancreatic tumor vasculature, each of said peptides or peptidomimetics comprising at least 5 contiguous amino acids of an amino acid sequence selected from CKGAKAR (SEQ ID NO:19), and VGVGEWSV (SEQ ID NO:35), or a conservative variant or peptidomimetic thereof, or wherein each of said peptides or peptidomimetics binds specifically to a cognate receptor for an amino acid sequence selected from CKGAKAR (SEQ ID NO:19) and VGVGEWSV (SEQ ID NO:35); or wherein said at least two peptides or peptidomimetics selectively home to premalignant and malignant vasculature, each of said peptides or peptidomimetics comprising at least 5 contiguous amino acids of the amino acid sequence FRVGVADV (SEQ ID NO:27), or a conservative variant or peptidomimetic thereof, or wherein each of said peptides or peptidomimetics binds specifically to a cognate receptor for the amino acid sequence FRVGVADV (SEQ ID NO:27). 76-93. (canceled) 